Comprehensive coordination chemistry II vol 8

catecholate substrate Figure 13a46 and deacetoxycephalosporin synthase DAOCS with its -ketoglutarate cofactor Figure 13b,47 both with a sixth site available for O2 binding.Figures13c and

Introduction to Volume 8

Since the publication of CCC (1987), bioinorganic chemistry has blossomed and matured as aninterdisciplinary field, which is surveyed in this volume from the perspective of coordinationchemistry Fully comprehensive coverage of biological inorganic chemistry is not possible, so asubset of topics is presented that captures the excitement of the field and reflects the scope anddiversity of the systems and research approaches used As an introduction, a summary ofstructural motifs that pervade bioinorganic systems is presented (Chapter 1) Subsequent chaptersfocus on the nature of the metal sites in proteins that participate in electron transfer (Chapters 2–4)and on the transport and storage of metal ions within the biological milieu (Chapters 5–9) Thediverse and biologically important array of metalloproteins that bind and activate dioxygen andperform oxidation reactions are then discussed (Chapters 10–18) To complete the presentation ofmetal–dioxygen chemistry, superoxide processing systems and photosynthetic oxygen evolutionare portrayed (Chapters 19–20) The following sections focus on the activation of other smallmolecules (H2, Chapter 21; N2, Chapter 22), mono- and dinuclear metal sites that performhydrolysis reactions (Chapters 23–24), and the burgeoning bio-organometallic area (Chapter 25).Proteins with synergistic metal–radical sites are discussed in Chapter 26 Iron–sulfur clusters arerevisited in Chapter 27, which presents those that are involved in enzyme catalysis rather thansimple electron transfer The role of metal ions in the environmentally significant process ofdenitrification is the focus of Chapter 28 Finally, the binding of metal ions to DNA and RNAare emphasized in Chapter 29 Together, the array of topics presented in this volume illustrates theimportance of coordination chemistry in the biological realm and the breadth of current bioinor-ganic chemistry research

L Que, Jr.Minnesota, USAMarch 2003

W B TolmanMinnesota, USAMarch 2003

xv

From Biology to Nanotechnology

Second Edition

Edited by

J.A McCleverty, University of Bristol, UK

T.J Meyer, Los Alamos National Laboratory, Los Alamos, USA

Description

This is the sequel of what has become a classic in the field, Comprehensive Coordination Chemistry The first edition, CCC-I, appeared in 1987 under the editorship of Sir Geoffrey Wilkinson (Editor-in-Chief), Robert D Gillard and Jon A McCleverty (Executive Editors) It was intended to give a contemporary overview of the field, providing both a convenient first source of information and a vehicle to stimulate further advances in the field The second edition, CCC-II, builds on the first and will survey developments since 1980 authoritatively and critically with a greater emphasis on current trends in biology, materials science and other areas of contemporary scientific interest Since the 1980s, an astonishing growth and specialisation of knowledge within coordination chemistry, including the rapid development of interdisciplinary fields has made it

impossible to provide a totally comprehensive review CCC-II provides its readers with reliable and informative background information in particular areas based on key primary and secondary references It gives a clear overview of the state-of-the-art research findings in those areas that the International Advisory Board, the Volume Editors, and the Editors-in-Chief believed to be especially important to the field CCC-II will provide researchers at all levels of sophistication, from academia, industry and national labs, with an unparalleled depth of coverage.

Bibliographic Information

10-Volume Set - Comprehensive Coordination Chemistry II

Hardbound, ISBN: 0-08-043748-6, 9500 pages

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Last update: 10 Sep 2005

Volume 1: Fundamentals: Ligands, Complexes, Synthesis, Purification, and StructureVolume 2: Fundamentals: Physical Methods, Theoretical Analysis, and Case StudiesVolume 3: Coordination Chemistry of the s, p, and f Metals

Volume 4: Transition Metal Groups 3 - 6

Volume 5: Transition Metal Groups 7 and 8

Volume 6: Transition Metal Groups 9 - 12

Volume 7: From the Molecular to the Nanoscale: Synthesis, Structure, and PropertiesVolume 8: Bio-coordination Chemistry

Volume 9: Applications of Coordination Chemistry

Volume 10: Cumulative Subject Index

10-Volume Set: Comprehensive Coordination Chemistry II

Recurring structural motifs in bioinorganic chemistry (L Que, W Tolman)

Electron transfer: Cytochromes (K Rodgers, G.S Lukat-Rodgers)

Electron transfer: Iron-Sulphur Clusters (R Holm)

Electron transfer: Cupredoxins (Yi Lu)

Alkali and alkaline earth ion recognition and transport (J.A Cowan)

Siderphores and transferrins (K.N Raymond, E.A Dertz))

Ferritins (A.K Powell)

Metal ion homeostasis (A.C Rosenzweig, R.L.Lieberman)

Metallothioneins (P Gonzalez-Duarte)

Dioxygen-binding proteins (D Kurtz)

Heme peroxidases (B Meunier)

Cytochrome P450 (Wonwoo Nam)

Non-heme Di-iron enzymes (S.J Lippard, Dongwhan Lee)

Non-heme mono-iron enzymes (J.P Caradonna, T.L Foster)

Dicopper enzymes (Shinobu Itoh)

Mono-copper oxygenases (M.A Halcrow)

Multi-metal oxidases (K.D Karlin et al.)

Molybdenum and Tungsten enzymes (C.D Garner et al.) Superoxide processing (A.F Miller)

NO chemistry (J.T Groves)

Oxygen evolution (G.W Brudvig, J Vrettos)

Hydrogen activation (M.Y Darensbourg, I.P Georgakaki)

Nitrogen fixation (P.L Holland)

Zinc hydrolases (E Kimura, Shin Aoki)

Dinuclear hydrolases (B.A Averill)

Bio-organometallic chemistry of cobalt and nickel (C.G Riordan)

Metal-radical arrays (W Tolman)

Iron sulfur clusters in enzyme catalysis (J.B Broderick)

Denitrification (R.R Eady, S.S Hasnain)

DNA and RNA as ligands (V.J DeRose et al.)

Reviews (University of Newcastle-Upon-Tyne, UK)

This impressive volume consisting of 29 articles from 45 different authors is an absolute must for all

those looking for an introduction, or already working in the area of Bio-coordination Chemistry

It is the most up-to-date and comprehensive account yet to appear conveying much of the excitement in a still rapidly expanding area.

Recurring Structural Motifs

in Bioinorganic Chemistry

L QUE, JR and W B TOLMAN

University of Minnesota, Minneapolis, Minnesota, USA

Perhapsthe most recognizable active-site motif in metalloproteinsisthe tetradentate porphyrinmacrocycle, the organic cofactor associated with heme proteins The most commonly encounteredisprotoporphyrin IX or heme b (Figure 1), found in the active sites of dioxygen-binding globins,dioxygen-activating cytochrome P450, H2O2-activating peroxidases, and b-type cytochromesinvolved in electron transfer (see Chapters 8.2, 8.10–8.12, and 8.17) Addition of cysteinylresiduesto the two vinyl groupson the porphyrin periphery convertsheme b to heme c (Figure 1),which are found in c-type cytochromesthat are also involved in electron transfer With fourequatorial sites of a potentially octahedral metal center occupied by this tetrapyrrole macrocycle,the chemistry of the iron can be modulated principally by changing the nature of the fifth andsixth ligands In electron-transfer proteins, both sites are occupied, usually by His/His or His/Metresidues, as in cytochromes b5 and c553,9respectively (Figure 2) For oxygen-binding globinsandheme enzymes, only the fifth (or proximal) site is occupied by a protein residue, leaving the sixthsite available for binding small molecules such as O2, CO, NO, or H2O2 The proximal ligand is

1

typically His(myoglobin,10 hemoglobin, horseradish peroxidase), Tyr (catalase11), or chrome P45012and chloroperoxidase) (Figure 3) The chemistry of the active site can be furtheraffected by second-sphere residues such as Glu or Gln that can hydrogen-bond to the proximalHis to modulate its basicity, or those on the distal side that can serve as acids and/or bases to aid

Cys(cyto-in the bCys(cyto-indCys(cyto-ing of dioxygen or the cleavage of the O
O bond The presence or absence of the lattercan in fact determine whether a dioxygen moiety in the active site becomes an electrophilicoxidant, asin the case of hydrocarbon-oxidizing cytochrome P450,13or actsasa nucleophile, as

in the case of the estradiol-producing enzyme aromatase.14The rich chemistry of heme proteinsand enzymes is discussed in Chapters 8.11, 8.12, and 8.17

There are other tetrapyrrole macrocyclesfound in nature besidesprotoporphyrin IX (Figure 1).Differences with the latter can be as simple as changes in the substituents on the porphyrinperiphery, such as in heme a and heme a3, which are key componentsof the mammalianrespiratory enzyme cytochrome oxidase (see Chapter 8.17) Other disparities can entail changes

in the oxidation level of the macrocycle, such as the two-electron reduced heme d of cytochrome

N

N

N

NFe

bd of E coli, and the four-electron reduced sirohemes of sulfite oxidase and nitrite reductase (seeChapter 8.28) Factor F430 isa highly reduced tetrapyrrole ligand that bindsnickel and isinvolved in methanogenesis, while cobalt-containing coenzyme B12 hasa corrin ring with onefewer carbon than in the macrocyle (see Chapter 8.25).

Another easily recognizable bioinorganic motif is the iron–sulfur cluster exemplified by the

Fe2S2rhomb and the Fe4S4cubane (Figure 4), most often found in ferredoxins involved in potential electron transfer (see Chapter 8.3).15 The iron ions in these clusters typically have adistorted tetrahedral geometry, with cysteine residues serving as terminal ligands The Fe2S2cluster (Figure 4a) cyclesbetween the þ2 and þ1 oxidation states, with redox potentials in the

low-
200 mV range.16A variation is the Rieske cluster found in the respiratory electron-transfer chainand some dioxygenases that operates in theþ200 mV range (Figure 4b).17In thiscluster, the twoterminal cysteinates on one iron are replaced by neutral histidine ligands, resulting in the upshift

N N

N His

His

O O

Fe O O

Cys

camphor

N N N

N

Fe N N

Ty r

N N

(c)

Figure 3 Structuresof the metal-containing portion of (a) oxymyoglobin (PDB 1A6M); (b) catalase (PDB1DGF); (c) the oxygen adduct of reduced cytochrome P450 with camphor substrate bound (PDB 1DZ4)

in potential The Fe4S4cluster (Figure 4c)18also typically cycles between theþ2 and þ1 oxidationstates, but can access the þ3 oxidation state in some cases and the 0 oxidation state in the Feprotein of nitrogenase.19Iron–sulfur clusters thus provide Nature with considerable flexibility inthe potentialsof electronsthey can transfer.

There are also cuboidal FeS clusters in which one of the iron sites is significantly different fromthe other three The extreme example isthe Fe3S4cluster, where one of the Fe corners is missing.Although originally considered to be an artifact of oxidative damage to iron–sulfur proteins, as inthe ferredoxin from Azotobacter vinelandii (Figure 5a),20 such clusters have been found in activeenzymes, e.g., the NiFe hydrogenase,21and are presumably involved in the electron-transfer chainneeded to deliver electrons to the heterodinuclear NiFe enzyme active site Aconitase is anotherenzyme with a site-differentiated Fe4S4cluster This key enzyme of the Krebs cycle catalyzes theisomerization of citrate to isocitrate (see Chapter 8.27) The isomerization occurs on one specific

Fe of the Fe4S4 cluster Instead of having a terminal Cys ligand, the unique Fe has a terminalaqua ligand in the resting state and binds the substrate, thereby increasing its coordinationnumber during the catalytic cycle (Figure 5b).22 Thus, the aconitase Fe4S4 cluster does notwork as an electron-transfer site in this enzyme, but instead provides a metal center that functionsasa Lewisacid There isalso recent evidence that an Fe4S4cluster can act both as an electron-transfer site and a Lewis acid center In S-adenosylmethionine-dependent iron–sulfur enzymes,one Fe of the cluster acts to bind the carboxylate of the S-adenosylmethionine cofactor prior tothe redox reaction (see Chapter 8.27)

One Fe isreplaced by another metal ion in other site-differentiated Fe4S4clusters Many ofthese examples derive from the chemical reconstitution of an Fe3S4cluster with another metal ion,e.g., ZnFe3S4, CoFe3S4, CdFe3S4, etc.15 In addition, CO dehydrogenase has been found tohave an NiFe S cluster that is presumably involved in CO binding and activation (Figure 5c)

Cys

(a)

(c)

Figure 4 Structuresof the (a) Fe2S2(Cys)4ferrodexin site (PDB 1AWD); (b) Fe2S2(Cys)2(His)2‘‘Rieske’’ site

(PDB 1JM1); (c) Fe4S4(Cys)4ferrodoxin site (PDB 2FDN)

(see Chapter 8.25).23 The novel cluster has a structure that significantly deviates from thepostulated model in which the Ni ion is appended to an intact Fe4S4cluster; instead, the Ni ion

is integrated into the cluster structure, which could be construed as arising from the coordination

of a (Cys)Ni--S(R)-Fe(Cys)(His) unit to three of four -S atomsof an Fe3S4cluster

The iron–sulfur clusters of the MoFe protein of nitrogenase illustrate another variation on thecuboidal M4S4 theme The P and M clusters of this protein can be formulated as vertex-sharedbicubane units, perhaps required because the fixation of dinitrogen is thought to occur in two-electron reduction steps (see Chapter 8.22) For the P cluster, which serves as an electron-storageand -transfer site, two Fe4S4clusters combine to share a common 6-S vertex (Figure 6a).24Onthe other hand, the M cluster, which is believed to be the locus of nitrogen-fixing activity, is acombination of an Fe4S3and an MoFe3S3cuboidal unit sharing a common, newly discovered 6-vertex, which cannot be a sulfur atom (Figure 6b).24 The electron density associated with thisatom identifiesit asa low Z atom, and a 6-N is mechanistically the most attractive possibility.The third recurring structural motif with non-amino-acid components found in metalloproteins isthe metal–dithiolene unit found in molybdenum- and tungsten-containing oxidases or dehydrogenases(see Chapter 8.18) The dithiolene is typically a pterin derivative (often with phosphate and/ornucleotide appendages) and coordinated to Mo or W in a 1:1 or 2:1 stoichiometry (Figure 7).25,26These units usually function in two-electron redox reactions (cf ‘‘oxo transfer’’), shuttling between

MIVand MVIoxidation states

The most recent addition to this group of motifs is the Fe(CO)x(CN)yfragment found in theactive sites of both NiFe and Fe-only hydrogenases (Figure 8).21,27Thisorganometallic fragmentisconnected to a Ni(Cys) unit in the NiFe enzyme via two thiolate bridges(Figure 8a) and to

Fe

Fe

FeS

S

S

SCys

H2O

S

His

CysFe

Cys

“X”

Ni

SCys

FeS

Cys

FeS

FeCysS

another organometallic Fe fragment via a dithiolate bridge in the Fe-only enzyme (Figure 8b).The unusual organometallic nature of this fragment suggests an important role for H2activation,but more work is required to establish the mechanisms of action for these fascinating enzymes(see Chapter 8.21).

The tetrahedral M(Cys)4 unit is a commonly found structure in metalloproteins Besides the Nicenter in NiFe hydrogenases (Figure 8a),21thismotif isalso found for M = Fe, Zn, and Cd AnFe(Cys)4site is present in rubredoxin (Figure 9a),28one of the earliest characterized iron–sulfurproteins It also is found in dinuclear superoxide reductases,29where it is proposed to serve as anelectron-storage site for the superoxide-reducing active site (see Chapter 8.19) The M(Cys)4unit is

a structural component in Zn-containing alcohol dehydrogenase30 and a fragment of the Zn/Cdclusters of metallothioneins (Figure 9b; see Chapter 8.9).31Variationsof thistetrahedral motif occur

in Zn-finger proteinswhere a Zn(His)2(Cys)2unit iscommonly found (Figure 9c).32

The trigonal Cu(Cys)(His)2unit isa recurring motif in cupredoxins, more commonly known as

‘‘blue’’ copper proteins, which are principally involved in electron transfer in the high potential

MoHis

FeS

S

FeN

Fe

SS

SS

SFeFe

Cys

FeS

S

FeS

Fe

Cys

FeCys

range (see Chapter 8.4) The presence of axial ligation and the strength of such interactions withthe copper center modulate the redox potential to provide the range observed for these proteins.33Asillustrated inFigure 10, the cupredoxin site can be trigonal (Figure 10a),34trigonal pyramidal

delocalized, mixed-valence dicopper(I,II) centersof cytochrome oxidase (Figure 10d)37 andnitrousoxide reductase,38 which may be construed as a dimeric derivative in which one His oneach Cu isreplaced by another ligand (see Chapter 8.17)

The facial M(Xaa)3 unit isanother versatile building block, which issuitable for a variety ofmetal ionsand accommodatestetrahedral, trigonal-bipyramidal, square-pyramidal, and octahedral

MoO

DMSO

Pterin dithiolene

Pterin dithiolene

Guanosine diphosphate (truncated)

Guanosine

diphosphate

(truncated)

OS

S

SS

H2O(OH–)

(a)

(b)

Figure 7 Structuresof the (a) MoVIsite of aldehyde oxidoreductase (PDB 1HLR); (b) DMSO adduct to the

MoIVsite of dimethylsulfoxide reductase (PDB 4DMR) The guanine portions of the pterin cofactors in (b)

are omitted for clarity

“X”

Fe

FeFeFe

S

S

SS

Cys

FeCys

Cys

CysCys

ZnCysCys

His

His

(c)

Figure 9 Structuresof the (a) rubredoxin Fe(Cys)4site (PDB 1BRF); (b) Zn2Cd(Cys)9metallothionein site

(PDB 4MT2); (c) Zn(His)(Cys) ‘‘zinc finger’’ site (PDB 1A1H)

geometries The M(His)3motif is found in the tetrahedral metal sites of several metalloenzymes, suchascarbonic anhydrase (ZnII,Figure 11a),39nitrite reductase (CuII,Figure 11b),40and cytochrome coxidase (Cu,Figure 11c).41The fourth position is used to activate the water nucleophile in carbonicanhydrase, to bind nitrite in nitrite reductase, and to serve as the initial binding site for O2(or CO) incytochrome c oxidase Thismotif also providesthe remaining ligandsof the square-pyramidal ionsofthe Cu2(-2:2-O2) core in oxyhemocyanin (Figure 12a)42 and one six-coordinate iron ion of thediiron site in oxyhemerythrin (Figure 12b).43

An additional recurring facial motif isthe 2-His-1-carboxylate triad found in a number ofmononuclear nonheme iron enzymesthat activate O2.44,45 Thistriad servesto hold the iron(II)center in the active site and provides three solvent-accessible sites to bind exogenous ligands Thissuperfamily of enzymes can catalyze a range of oxidative transformations, including the cis-dihydroxylation of arene double bondsand the oxidative cleavage of catecholsin the biodegrad-ation of aromatics, the formation of the -lactam and thiazolidine ringsof penicillin, thehydroxylation of Phe, Tyr, and Trp with the help of a tetrahydrobiopterin cofactor, and theoxidative decarboxylation of an -ketoglutarate co-substrate to generate an oxidant capable offunctionalizing C
H bonds (see Chapter 8.14) The coordinative versatility of this active-sitemotif isillustrated in Figure 13 Figures13a and 13b show the active-site structures of binaryenzyme–bidentate substrate complexes, an extradiol cleaving catechol dioxygenase with its

Cu

Cu

MetCys

catecholate substrate (Figure 13a)46 and deacetoxycephalosporin synthase (DAOCS) with its-ketoglutarate cofactor (Figure 13b),47 both with a sixth site available for O2 binding.Figures13c and13dshow the active sites of ternary complexes: isopenicillin N synthase (IPNS) with thecoordinated thiolate of its tripeptide substrate and NO bound as a surrogate for O 48 and

H2O

CuHis

Tyr

His

His

FeN

N

Figure 11 Structures of the active sites of (a) carbonic anhydrase (PDB 1CA2); (b) nitrite reductase (PDB

2NRD); (c) cytochrome c oxidase (heme a3-CuBpair; PDB 1OCR)

Cu O

OCuHis

His

HisHis

OH

HisFe

Figure 12 Structuresof the active sitesof (a) oxyhemocyanin (PDB 1OXY); (b) oxyhemerythrin (PDB

1HMO)

naphthalene dioxygenase with a side-on bound dioxygen,49 presumably poised to effect the dihydroxylation of a double bond on the nearby arene substrate (not shown) These examplesillustrate how a diversity of reactions can be obtained from the flexibility of the 2-His-1-carbox-ylate motif, which enables the iron(II) center to bind and activate substrates, cofactors and/or O2.Other combinations of histidine and carboxylate ligands also can be found in a number ofmetalloenzymes Quercetinase is a copper enzyme that uses a 3-His-1-carboxylate combinationarranged in a square-pyramidal geometry, with one basal site available for the coordination ofsubstrate (Figure 14a).50Fe and Mn superoxide dismutases utilize a 3-His-1-carboxylate combin-ation in a sawhorse arrangement to provide coordination sites for two exogenous ligands (Figure14b).51 These coordination environments can in fact be construed as combinations of M(His)3and M(His)2(carboxylate) triads Alternatively, some dimetal hydrolases like the -lactamasefrom S maltophilia (see Chapter 8.24) combine the two triad types via a hydroxo bridge togenerate a (His)3Zn--OH-Zn(His)2(Asp) active site (Figure 14c).52Note how, in most examples,the monodentate carboxylate ligand hydrogen bondsto a bound water or hydroxide.

cis-A number of other metalloenzymeshave MII2(His)2(O2CR)4 active sites (see Chapter 8.13).Most prominent of these are the di-iron enzymes, including the hydroxylase component ofmethane monooxygenase (MMOH, Figure 15a)53 and class 1 ribonucleotide reductase R2proteins(Figure 15b).54 These enzymes have two conserved Asp/Glu(Xaa)nGluXaaXaaHissequence motifs in a four-helix bundle that provide the six amino-acid ligands for the di-iron

(substrate)

His

H2OGlu

(d)

FeHis

with bound dioxygen in the presence of substrate (not shown) (PDB 1O7M)

active site The di-iron center activates O2and carriesout the hydroxylation of alkanesand theformation of a catalytically essential tyrosyl radical for the conversion of ribonucleotides todeoxyribonucleotides, respectively The carboxylates more distant in sequence from the othertwo residues act as terminal ligands, while the two near in sequence to the His ligands act tobridge the iron atoms, with both monodentate and bidentate modes observed Upon oxidation ofthese di-iron(II) enzymes to the di-iron(III) state, there is a change in core structure, resulting inthe shift of one carboxylate bridge to a terminal position (the so-called carboxylate shift55) and theintroduction of an oxo bridge or two hydroxo bridgesto neutralize the Lewisacidity of the iron(III)ions(Figures15cand15d).53,56(An oxo bridge isalso observed in the oxidized form of an unrelateddiiron protein hemerythrin (Figure 12b).) Related dinuclear active sites are found for fatty aciddesaturases,57rubrerythrin,58the ferroxidase site in ferritins,59and the dimanganese catalase.60The crystal structures of nitrile hydratase and acetyl CoA synthase both show a metal centercoordinated to a planar N2S2unit derived from a CysXaaCys tripeptide, with the nitrogen ligandsarising from the peptide amidates of the Xaa residue and the latter Cys residue These resultsemphasize a point originally made in the explorations of the coordination chemistry of peptideligands: that the peptide nitrogen can bind to metal centers, particularly in its anionic form In therecently reported structures of acetyl CoA synthase, there is a planar Ni(N2S2) unit derived from

a Cys595Gly596Cys597 tripeptide segment (Figure 16a).61,62 Thisunit isin turn connected to an

Fe4S4 cluster via an intervening metal ion, which can be Ni, Cu, or Zn in the three structuresreported In nitrile hydratase, the low-spin iron(III) ion is coordinated to the planar N2S2unitfrom Cys112Ser113Cys114and additionally ligated by an axial thiolate from nearby Cys109(Figure16b).63 The sixth site can be occupied by a solvent molecule, which presumably is involved in

Zn

Figure 14 Structures of the active sites of (a) quercetinase (PDB 1JUH); (b) Mn superoxide dismutase (PDB

3MDS); (c) a dizinc -lactamase (PDB 1SML)

nitrile hydrolysis, or NO, which is an inhibitor Further making this active-site structure unique isthe observed post-translational oxidation of the thiolates of Cys112 and Cys114 to a sulfinate(RSO
) and a sulfenate (RSO
), respectively, required for enzyme activity.

GluGlu

Fe

His

HisFe

Figure 15 Structuresof the di-iron active sitesof (a) reduced MMOH (PDB 1FYZ); (b) reduced tide reductase (PDB 1XIK); (c) oxidized MMOH (PDB 1FZ1); (d) oxidized ribonucleotide reductase (PDB

ribonucleo-1AV8)

(b) (a)

SCu

peptideligandS

SNi

NO

O

NON

O

peptideligand

Figure 16 Structures of (a) the multimetallic site of acetyl CoA synthase (PDB 1MJG); (b) the active site of

the NO adduct of nitrile hydratase (PDB 2AHJ)

As a final example, we note the similarity of the square-pyramidal FeN4(Cysaxial) site found insuperoxide reductase29(Figure 17) to those of cytochrome P450 (Figure 3c) and chloroperoxidase.While the N4 unitsin cytochrome P450 and chloroperoxidase derive from a porphyrin, the fournitrogens of superoxide reductase are from four His residues Interestingly, this motif is used inthe two heme enzymesto bind and activate dioxygen moietiesto generate a high-valent oxoironspecies capable of substrate oxidations, while the corresponding site in superoxide reductase isused to protect anaerobes from the toxic effects of superoxide by reducing it to H2O2.

In this introductory overview, we have attempted to point out structural similarities amongmetalloprotein active sites to emphasize how Nature has utilized coordination chemistry to heradvantage, particularly in generating related active sites with different functions In highlightingcommon features, we have necessarily de-emphasized unique sites such as the unusual Cu4(4-S)cluster of nitrous oxide reductase (see Chapter 8.28) and the critical Mn4 cluster in the oxygen-evolving complex of photosynthesis (see Chapter 8.20), whose precise structure is still developing

as higher resolution crystallographic data become available Also excluded are possible motifsthat may emerge in the areas of metal-ion transport and homeostasis (see Chapters 8.5, 8.6, and8.8) and metal ion/nucleic acid interactions(see Chapter 8.28) It isour hope that thisoverviewprovides a sufficient basis for underscoring the importance of coordination chemistry inbioinorganic chemistry

ACKNOWLEDGMENT

We thank John York for his assistance with generating the figures in this article

1 Lippard, S J.; Berg, J M Principles of Bioinorganic Chemistry; University Science: Mill Valley, CA, 1994.

2 Kaim, W.; Schwederski, B Bioinorganic Chemistry: Inorganic Elements in the Chemistry of Life; Wiley: New York, 1991.

3 Bertini, I.; Gray, H B.; Lippard, S J.; Valentine, J S., Eds Bioinorganic Chemistry; University Science Books: Mill Valley, CA, 1994.

4 Frau´sto da Silva, J J R.; Williams, R J P The Biological Chemistry of the Elements: The Inorganic Chemistry of Life; Clarendon Press: Oxford, UK, 1991.

5 Cowan, J A Inorganic Biochemistry: An Introduction; 2nd ed.; Wiley-VCH: New York, 1997.

6 Holm, R H.; Solomon, E I Chem Rev 1996, 96, 2239–3042, entire issue, edited by Holm Solomon.

7 Holm, R H.; Kennepohl, P.; Solomon, E I Chem Rev 1996, 96, 2239–2314.

8 Durley, R C E.; Mathews, F S Acta Crystallogr D Biol Crystallogr 1996, 52, 65–66.

9 Benini, S.; Gonzalez, A.; Rypniewski, W R.; Wilson, K S.; Van-Beeumen, J J.; Ciurli, S Biochemistry 2000, 39, 13115–13126.

10 Vojtechovsky, J.; Chu, K.; Berendzen, J.; Sweet, R M.; Schlichting, I Biophys J 1999, 77, 2153–2174.

11 Putnam, C D.; Arvai, A S.; Bourne, Y.; Tainer, J A J Mol Biol 2000, 296, 295–309.

12 Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A M.; Maves, S A.; Benson, D E.; Sweet, R M.; Ringe, D.; Petsko, G A.; Sligar, S G Science 2000, 287, 1615–1622.

Fe

CysHis

His

Glu

HisHis

Figure 17 Structure of the catalytic active site of superoxide reductase (PDB 1DQI)

13 Ortiz de Montellano, P R., Ed Cytochrome P450: Structure, Mechanism, and Biochemistry; 2nd ed.; Plenum: New York, 1995.

14 Wertz, D L.; Valentine, J S Struct Bonding 2000, 97, 38–60.

15 Beinert, H.; Holm, R H.; Mu¨nck, E Science 1997, 277, 653–659.

16 Bes, M T.; Parisini, E.; Inda, L A.; Saraiva, L.; Peleato, M L.; Sheldrick, G M to be published (PDB 2FDN).

17 Boenisch, H.; Schmidt, C L.; Schaefer, G.; Ladenstein, R J Mol Biol 2002, 319, 791–805.

18 Dauter, Z.; Wilson, K S.; Sieker, L C.; Meyer, J.; Moulis, J M Biochemistry 1997, 36, 16065–16073.

19 Yoo, S J.; Angove, H C.; Burgess, B K.; Hendrich, M P.; Mu¨nck, E J Am Chem Soc 1999, 121, 2534–2545.

20 Schipke, C G.; Goodin, D B.; McRee, D E.; Stout, C D Biochemistry 1999, 38, 8228–8239.

21 Volbeda, A.; Garcia, E.; Piras, C.; deLacey, A L.; Fernandez, V M.; Hatchikian, E C.; Frey, M.; Fontecilla Camps,

J C J Am Chem Soc 1996, 118, 12989–12996.

22 Lauble, H.; Kennedy, M C.; Beinert, H.; Stout, C D J Mol Biol 1994, 237, 437–451.

23 Drennan, C L.; Heo, J.; Sintchak, M D.; Schreiter, E.; Ludden, P W Prod Nat Acad Sci USA 2001, 98, 11973–11978.

24 Eins le, O.; Tezcan, F A.; Andrade, S L A.; Schmid, B.; Yos hida, M.; Howard, J B.; Rees , D C Science 2002, 297, 1696–1700.

25 Rebelo, J M.; Dias, J M.; Huber, R.; Moura, J J.; Ramao, M J J Biol Inorg Chem 2001, 6, 791–800.

26 McAlpine, A S.; McEwan, A G.; Bailey, S J Mol Biol 1998, 275, 613–623.

27 Nicolet, Y.; Piras, C.; Legrand, P.; Hatchikian, C E.; Fontecilla-Camps, J C Structure Fold Des 1999, 7, 13–23.

28 Bau, R.; Rees, D C.; Kurtz, D M.; Scott, R A.; Huang, H S.; Adams, M W W.; Eidsness, M K J Biol Inorg Chem 1998, 3, 484–493.

29 Yeh, A P.; Hu, Y.; Jenney, F E., Jr.; Adams, M W W.; Rees, D C Biochemistry 2000, 39, 2499–2508.

30 Eklund, H.; Samma, J P.; Wallen, L.; Branden, C I.; Akeson, A.; Jones, T A J Mol Biol 1981, 146, 561–587.

31 Braun, W.; Vasak, M.; Robbins, A H.; Stout, C D.; Wagner, G.; Kagi, J H.; Wuthrich, K Proc Natl Acad Sci USA 1992, 89, 10124–10128.

32 Elrod-Erickson, M.; Benson, T E.; Pabo, C O Structure 1998, 6, 451–464.

33 Gray, H B.; Malmstrom, B G.; Williams, R J P J Biol Inorg Chem 2000, 5, 551–559.

34 Ducros, V.; Brzozowski, A M.; Wilson, K S.; Ostergaard, P.; Schneider, P.; Svendson, A.; Davies, G J Acta Crystallogr D 2001, 57, 333–336.

35 Kohzuma, T.; Inoue, T.; Yoshizaki, F.; Sasakawa, Y.; Onodera, K.; Nagatomo, S.; Kitagawa, T.; Uzawa, S.; Isobe, Y.; Sugimura, Y.; Gotowda, M.; Kai, Y J Biol Chem 1999, 274, 11817–11823.

36 Chen, Z W.; Barber, M J.; McIntire, W S.; Mathews, F S Acta Crystallogr D 1998, D54, 253–268.

37 Williams, P A.; Blackburn, N J.; Sanders, D.; Bellamy, H.; Stura, E A.; Fee, J A.; McRee, D E Nat Struct Biol.

1999, 6, 509–516.

38 Brown, K.; Tegon, M.; Prudencio, M.; Pereira, A S.; Besson, S.; Moura, J J.; Moura, I.; Cambillau, C Nat Struct Biol 2000, 7, 191–195.

39 Eriksson, A E.; Jones, T A.; Liljas, A Proteins 1988, 4, 274–282.

40 Adman, E T.; Godden, J W.; Turley, S J Biol Chem 1995, 270, 27458–27474.

41 Yoshikawa, S.; Shinzawa-Itoh, K.; Nakashima, R.; Yaono, R.; Yamashita, E.; Inoue, N.; Yao, M.; Fei, M J.; Libeu,

C P.; Mizushima, T.; Yamaguchi, H.; Tomizake, T.; Tsukihara, T Science 1998, 280, 1723–1729.

42 Magnus, K A.; Hazes, B.; Ton-That, H.; Bonaventura, C.; Bonaventura, J.; Hol, W G J Proteins 1994, 19, 302–309.

43 Holmes, M A.; Le Trong, I.; Turley, S.; Sieker, L C.; Stenkamp, R E J Mol Biol 1991, 218, 583–593.

44 Hegg, E L.; Que, L., Jr Eur J Biochem 1997, 250, 625–629.

45 Que, L., Jr Nat Struct Biol 2000, 7, 182–184.

46 Vaillancourt, F H.; Han, S.; Fortin, P D.; Bolin, J T.; Eltis, L D J Biol Chem 1998, 273, 34887–34895.

47 Valegard, K.; van Scheltinga, A C.; Lloyd, M D.; Hara, T.; Ramaswamy, S.; Perrakis, A.; Thompson, A.; Lee, H J.; Baldwin, J E.; Schofield, C J.; Hajdu, J.; Andersson, I Nature 1998, 394, 805–809.

48 Roach, P L.; Clifton, I J.; Hens gens , C M.; Shibata, N.; Schofield, C J.; Hajdu, J.; Baldwin, J E Nature 1997, 387, 827–830.

49 Karlsson, A.; Parales, J V.; Parales, R E.; Gibson, D T.; Eklund, H.; Ramaswamy, S Science 2003, 299, 1039–1042.

50 Fusetti, F.; Schroeter, K H.; Steiner, R A.; Van Noort, P I.; Pijning, T.; Rozeboom, H J.; Kalk, K H.; Egmond, M R.; Dijkstra, B W Structure 2002, 10, 259–268.

51 Ludwig, M L.; Metzger, A L.; Pattridge, K A.; Stallings, W C J Mol Biol 1991, 219, 335–358.

52 Ullah, J H.; Walsh, T R.; Taylor, I A.; Emery, D C.; Verma, C S.; Gamblin, S J.; Spencer, J J Mol Biol 1998,

284, 125–136.

53 Whittington, D A.; Lippard, S J J Am Chem Soc 2001, 123, 827–836.

54 Logan, D T.; Su, X D.; Aberg, A.; Regnstrom, K.; Hajdu, J.; Eklund, H.; Nordlund, P Structure 1996, 4, 1053–1064.

55 Rardin, R L.; Tolman, W B.; Lippard, S J New J Chem 1991, 15, 417–430.

56 Tong, W.; Burdi, D.; Riggs-Gelasco, P.; Chen, S.; Edmondson, D.; Huynh, B H.; Stubbe, J.; Han, S.; Arvai, A.; Tainer, J Biochemistry 1998, 37, 5840–5848.

57 Lindqvist, Y.; Huang, W.; Schneider, G.; Shanklin, J EMBO J 1996, 15, 4081–4092.

58 Jin, S.; Kurtz, D M., Jr.; Liu, Z.-J.; Rose, J.; Wang, B.-C J Am Chem Soc 2002, 124, 9845–9855.

59 Stillman, T J.; Hempstead, P D.; Artymiuk, P J.; Andrews, S C.; Hudson, A J.; Treffry, A.; Guest, J R.; Harrison,

P M J Mol Biol 2001, 307, 587–603.

60 Whittaker, M M.; Barynin, V V.; Antonyuk, S V.; Whittaker, J W Biochemistry 1999, 38, 9126–9136.

61 Doukov, T I.; Iverson, T M.; Seravalli, J.; Ragsdale, S W.; Drennan, C L Science 2002, 298, 567–572.

62 Darnault, C.; Volbeda, A.; Kim, E J.; Legrand, P.; Verne`de, X.; Lindahl, P A.; Fontecilla-Camps, J C Nat Struct Biol 2003, 271–279.

63 Nagashima, S.; Nakasako, M.; Dohmae, N.; Tsujimura, M.; Takio, K.; Odaka, M.; Yohda, M.; Kamiya, N.; Endo, I Nat Struct Biol 1998, 5, 347–351.

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Comprehensive Coordination Chemistry II

ISBN (set): 0-08-0437486 Volume 8, (ISBN 0-08-0443303); pp 1–15

Electron Transfer: Cytochromes

K R RODGERS and G S LUKAT-RODGERS

North Dakota State University, Fargo, USA

8.2.2 HEME ELECTRONIC STRUCTURE AND AXIAL-LIGAND GEOMETRIES 20 8.2.2.1 Structural Aspects of 6cLS Iron Porphyrinates 20

8.2.2.3 The Frontier Orbitals and Fe–Ligand Bonding 21

8.2.4.3 Axial Heme Ligand Mutants of Cytochrome b5 48

17

Cross-linking of thehemeb vinyl groups at -pyrrolepositions 2 and 4, with Cys sidechains,covalently links the heme to the proteins in c-typecytochromes Cross-linking occurs at highlyconserved –Cys–Xxx–Yyy–Cys–His– sequences In heme a, hydroxyl and farnesyl groups areadded to the vinyl side chain at position 2, with a formyl group at position 8 Heme a is found

in proteins like cytochrome c oxidase Heme d1contains ketone groups in place of the position 2and 4 vinyl groups In cytochrome cd1nitrite reductase, the d-type cytochrome has been identified

as 3,8-dioxo-17-acrylate-porphyrindione.4 As this chapter will focus on electron-transfer chromes, a subset of the proteins containing porphyrin-based heme complexes, the discussion ofthe interplay between coordination chemistry and electronic structure will focus on their porphy-rinate ligands and their iron complexes

cyto-N

NN

NFe

OH

O

HOO

N

NN

NFe

OH

O

HOO

HO

HO

N

NN

NFe

OH

O

HOO

S

S

N

NN

NFe

OH

O

HOO

OHO

O

OHO

O

N

NN

NFe

OH

OH

O

HOOOH

I/A II/B

III/C IV/D

counterclockwise) from that shown here

Electron-transfer cytochromes are, with one exception (cytchromes c0), six-coordinate, low-spin(6cLS) with axial ligands being supplied by Lewis-basic amino-acid side chains or, less commonly,N-terminal amine groups The known axial ligand pairs are illustrated in Figure2 In b-typecytochromes, the heme is bound to the protein via two axial ligands of the heme iron, usuallyhistidines (His) A His residue is also one of the axial ligands in c-typecytochromes It is found in theconserved –Cys–Xxx–Yyy–Cys–His– sequence, which also anchors the porphyrin macrocycle to theprotein In c-type cytochromes, the most common second axial ligand for the heme iron isthe thioether side chain of a methionine (Met) However, bis-His heme coordination is knownand occurs most frequently in multiheme c-typecytochromes.5TheHis ligands arecoordinated totheiron through theN", although there is at least one example of N coordination.6In theuniquecaseof cyt f, a c-typecytochromein thephotosynthetic cyt b6fcomplex, the heme iron is coordinated

by a His residueand theamineof theN-terminal tyrosine(Tyr).7Finally, theiron-storageprotein,bacterioferritin, contains a b-type cytochrome thought to be involved in redox-coupled release ofiron from the bacterioferritin iron core.8This cytochromeexhibits bis-Met axial ligation, with thethioether ligands being supplied by symmetry-related pairs of bacterio ferritin subunits.9,10

cyto-out-of-planedistortion

8.2.2 HEME ELECTRONIC STRUCTURE AND AXIAL-LIGAND GEOMETRIES

8.2.2.1 Structural Aspects of 6cLS Iron Porphyrinates

In early studies of cytochromes, spectroscopic methods were relied upon for determination of spinstate, coordination number, and axial-heme ligation Model hemes in which metal-ion size,charge, and d-orbital occupancy, as well as ligand structure and composition, could be system-atically varied, were spectrally characterized for comparison to cytochromes Synthetic 6cLS FeIIand FeIII porphyrinates have been studied for many years with the goal of clarifying structure–function relationships observed in cytochromes

Thestructures of many 6cLS FeIIand FeIIIporphyrinates are available.11 TheFeIIIcomplexesexhibit several characteristic structural properties There is little or no displacement (0.09 A˚) of the

FeIIIcenter from the mean heme plane, even in complexes with different axial ligands The Fe–

Nprrole(FeNp) bond lengths depend upon the charge of the complex, with FeNpbeing1.990 A˚for neutral or positively charged complexes, and 2.000 A˚ for complexes with a single negativecharge.11 TheFeNp distances within a given complex can be inequivalent if axial ligands arecoplanar and nearly aligned with the FeNp bonds ( 0,Figure3), thoseperpendicular to theligand plane being shorter by0.02 A˚ Axial FeIIIL bond lengths depend upon orientation if theligand is planar Steric interactions of axial ligands like pyridines and hindered imidazoles with theporphyrin ring tend to lengthen the FeIIIL bond Finally, -acceptor ligands can induce ruffling ofthe porphyrin core by electronic stabilization

Equatorial FeNp bond lengths in the 6cLS FeII porphyrinates are only slightly longer(0.0102 A˚) than their FeIIIcounterparts, and in the complexes with identical axial ligands, the

FeII atom is in theplaneof theporphyrin core.11 In theLS d6 configuration of theferrouscytochromes and model hemes, filling of the d orbitals is expected to favor increased -bonding

in these complexes This is borne out by shortening of bonds with -acceptor ligands byhundredths of angstrom relative to the corresponding FeIII complexes.11 In model hemes, the

FeS bond lengths for neutral sulfur ligands appear to be insensitive to whether the iron is in theþ2 or þ3 oxidation state Bonds to neutral nitrogen donor ligands such as imidazoles, pyrazoles,and pyridines exhibit longer bonds in the FeIIcomplexes.11

8.2.2.2 Out-of-plane Porphyrin Deformation

One of the more extensively studied and discussed structural variables has been equilibrium of-planedistortion of theporphyrin Out-of-planeporphyrin deformation typically results in abathochromic (red) shift of the optical absorption spectrum and a shift to more negative reduc-tion potentials (easier to oxidize).12–15These tantalizing correlations, based upon studies of modelcomplexes comprising porphyrin ligands such as octaethyltetraphenylporphyrinate (OETPP2),having sterically crowded peripheries that force large out-of-plane deformations, have driven

N N

widespread investigation of the relationships between out-of-plane deformation, redox potential,and optical spectra of porphyrins and metalloporphyrins.12–14

Several reports have called into question the correlation between sterically enforced, of-plane deformation and the electronic properties, redox potential and spectroscopicsignatures.16–18 Based on UV–visible spectra, structural analysis, and TD-DFT calculations, ithas been suggested that the shifts in UV–visible transitions result from electronic effects of theperipheral substituents on the aromatic porphyrin core These effects drive a bond-alternatingrearrangement of the porphyrin core atoms called ‘‘in-plane nuclear reorganization.’’16

out-Regardless of the driving force(s), out-of-plane porphyrin deformations invariably occur alonglow-frequency, normal coordinates A recent computational method, normal-coordinate struc-tural decomposition (NSD), yields linear combinations of equilibrium distortions along the lowest-frequency out-of-plane normal vibrational coordinates of the heme.15,19,22 Thesix out-of-planevibrational coordinates of a D4hmetalloporphyrin used in the linear combinations are illustrated

distortions of nearly 1 A˚ along the ruf and/or sad coordinates, and it has been estimated that thesedistortions require heme protein interaction energies substantially greater than 2 kcal mol11and

up to 8 kcal mol11,23,24 Interestingly, out-of-plane deformations are largely conserved withinclasses of cytochromes.21,22,25,26 It has been suggested that expenditure of energy by a protein

to enforce a particular out-of-plane distortion is unlikely to be accidental, and could play animportant rolein biological function.15,23

8.2.2.3 The Frontier Orbitals and Fe–Ligand Bonding

porphyrin molecular orbitals (MOs)27 and the energy ordering and symmetry designations of

on opposite sides of the mean heme plane Adapted from ref.11

metal-centered d-orbitals in octahedral and tetragonal ligand fields, along with the frontier MOs

of the porphyrin Sigma bonding between iron and the porphyrin is based on interactions betweenthe dz2 (a1g in D4h) and dx2 y 2 (b1g in D4h) orbitals of iron and porphyrin MOs with respectivesymmetries The degenerate dxz, dyzpair (egin D4h) can combinewith theporphyrin  MOs of egsymmetry.27The dxyorbital is nonbonding in D4hmetalloporphyrins Of particular interest is thebonding interaction between the dxyorbital (b2symmetry) and the porphyrinate b2MO (correl-ates with the a2uMO in D4hcomplexes) in hemes of D2dsymmetry.24,27–32Symmetry lowering to

D2dis usually associated with the ruffling distortion shown inFigure4 This deformation involvescounterrotation of adjacent pyrrole rings about their FeNp bonds, which results in a nonzeroprojection of pyrrole N pz-derived MOs onto the mean porphyrin plane The resulting –dxybonding represents an electronic contribution to the stability of some ruffled 6cLS FeIIIporphy-rinates with (dxz,dyz)4(dxy)1 FeIII ground states This stabilization is not possible for FeII com-plexes, because the dxyorbital is filled

Sigma bonding between Fe and axial ligands generally involves 3dz 2, 4s, and 4pzorbitals of themetal and occupied orbitals of the axial ligands Axial ligands can also participate in  bondingwith FeIIIand FeIIcenters by interaction of their  orbitals with the partially or completely filled

dxzand dyzorbitals.27

In most 6cLS FeIIIhemes, the ordering is that of the (dxy)2(dxz,dyz)3ground state.27This stateissubject to stabilization by Jahn–Teller splitting of the egset, which results in symmetry loweringfrom fourfold to twofold rotational symmetry The stabilization due to Jahn–Teller splitting in6cLS FeIII porphyrinates can manifest itself as experimentally discernible symmetry lowering,including the thermodynamic preference for parallel orientations of planar axial ligands andasymmetry in FeNp bond lengths when the ligands align with two opposing FeNpbonds.11,25,33,34 Such complexes give rise to rhombic (type II) EPR spectra,27,28,35 as shown in

N N

-accepting axial ligand field

Nodal patterns & symmetries

match or mismatch of metal d- and porphyrin -orbitals

6cLS FeIIIhemes, the effects of Jahn–Teller splitting can be masked by strong steric interactionsbetween the porphyrin and axial ligands, due either to out-of-plane deformation of the porphyrin

or to sterically demanding axial ligands11such as 2-methylimidazole, which force the axial ligands

to be mutually perpendicular These complexes also have (dxy)2(dxz,dyz)3ground states and giveriseto highly anisotropic (typeI or ‘‘gmax’’) EPR spectra.27,28,35In complexes with strong axial-ligand fields imposed by weak -donor and/or -acceptor ligands, such as 4-cyanopyridine andtheorganic isocyanides, theMOs having contributions from thedxzand dyzAOs arestabilized.These complexes have axial (dxz,dyz)4(dxy)1ground states,11,27 which arenot susceptibleto Jahn–Teller distortion and exhibit type III EPR spectra.27 This ground state is further evidenced bysubstantial weakening of the porphyrin d (2B2!2

A1) NIR-CT MCD transition, which issymmetry forbidden for (dxz,dyz)4(dxy)1ground states.28

8.2.2.4 Cause and Effect Roles of Axial Ligation

Planar axial ligands can adopt mutually parallel or mutally perpendicular orientations in 6cLS

FeIII complexes, with their orientations relative to the FeN bonds ranging from 0 to 45.27

such as isocyanides Figure adapted from reference27

Relevant angles are shown in Figure3 There are two principal aspects of axial ligation toconsider, steric and electronic Planar axial ligands prefer mutually orthogonal orientations if

FeL bonding causes steric interactions between the axial ligands and the porphyrin Theseinteractions are stronger for the six-membered pyridine and sterically hindered 2-methlyimidazoleligands than for imidazole.11,27 They can involve atoms in the porphyrin core and/or bulkyperipheral substituents, and usually result in ruffling of the porphyrin core (see Figure4)

A distinct situation arises in persubstituted porphyrins, wherein strong steric interactionsoccur between adjacent substituents at the porphyrin periphery These porphryins exhibit aninherent saddling deformation, even as metal-free macrocycles, and exhibit such narrow cleftsthat steric constraints dictate orthogonal, or nearly orthogonal, orientations of axial ligands.27,36Most orthogonal axial-ligand orientations in 6cLS FeIII porphyrinates are attributable to therelief of costly intramolecular steric interactions.11,34,37–39This is an important point with regard

to the heme structures and deformations in cytochromes, as their hemes exhibit little intrinsicintramolecular steric strain.23 Furthermore, since all type b, c, and f cytochromes characterized

to datehave(dxy)2(dxz,dyz)3 ground states,27,40,41 there is no electronic stabilization of theruffling distortion as described above in these cytochromes Hence, equilibrium deviationsfrom planarity must be attributable to exogenous forces brought to bear on the heme by theprotein

In the absence of steric encumbrance at the porphyrin periphery, the parallel axial-ligandconformation is favored for ligands such as unhindered imidazoles This is attributed to stabiliza-tion of the Jahn–Teller distorted ground state.11,27,34 In these complexes, the porphyrin isinvariably flat and , theligand angle, can rangefrom 0 to 45 (Figure3) The complexeshaving  near 0 exhibit rhombic EPR spectra and a B1g-distorted porphyrin core (nonequivalentadjacent FeIIINp bond lengths) Those with  near 45 exhibit equivalent FeIIINp bondlengths, but still yield rhombic EPR spectra wherein the rhombicity, V (see Figure5), has beenshown to track inversely with .42Six-coordinate, low-spin FeIIcomplexes strongly prefer parallelaxial ligands and a planar porphyrin ligand.11,27,29This is thought to bebecauseof theinability oflow-spin FeIIto stabilize a ruffled porphyrin conformation Several examples of ruffled 6cLS FeIIcomplexes have been reported.43 Ruffling has been ascribed to steric interactions between bulkyporphyrin substituents and axial ligands Some work has suggested that FeNax bonding isindependent of -accepting ability of the axial ligands; these results were interpreted to meanthat dxz- and dyz-orbitals arenot strongly involved in -bonding.29Consistent with this reasoning,

a series of 4-substituted pyridines with varying pKas and -acceptor abilities show the samethermodynamic stabilities, suggesting that axial bond strengths do not vary significantly in thisseries This is in contrast to the analogous FeIIIcomplexes, wherein the FeNax bond strengthvaries predictably.44 Since the redox potential depends upon the ratio of FeIIIand FeIIstabilityconstants, log(2III/2II),44 tunability of the contribution of bond strengths to redox potentialseems to be confined to tunability of 2III in model complexes It is worth noting that this maynot be the case in cytochromes, because the proteins have control over porphyrin conformationand axial-ligand orientation, which can differ from the lowest-energy conformations of the modelcomplexes where there are no exogenous forces

8.2.3.1 Function

Cyt c has an important role in the production of ATP; in the mitochondrial respiratory transfer chain, cyt c transfers electrons from the transmembrane cyt bc1complex to cytochrome coxidase.45,46 Cyt c also delivers electrons to cytochrome c peroxidase, which facilitates thereduction of hydrogen peroxide to water In addition to its life-sustaining electron-transferfunctions, cyt c is required for activation of the cell-death protease, caspase-3, in apoptosis.47–49Defects in cyt c biogensis have been implicated in pathogenic responses related to copper50 andiron metabolism,51and prokaryotic heme biosynthesis.52

electron-Similar c-type cytochromes are involved in many kinds of energy metabolism in bacteria, such

as phototrophes, methylotrophes, sulfate reducers, nitrogen-fixers, and denitrifiers For example,

in the anaerobic electron chain of the denitrification system in Gram-negative bacteria, c-typecytochromes transfer electrons from the cyt bc complex to cytochrome cd nitrite reductase, NO

reductase, and NO reductase.53In plants and cyanobacteria, c-type cytochromes shuttle electronsfrom thecyt b6fcomplex to photosystem I.54

Dueto its central rolein thevital processes of living organisms, and thelargedatabaseofphysical and biochemical information available on cyt c, it has becomeoneof theparadigms

in the study of biological electron-transfer processes.55,56 Functional studies on cyt c andnumerous cyt c point mutants have been used to identify residues and regions of the proteinthat influence electron-transfer properties.55–57In order to study intraprotein electron transferand thepathways involved in cyt c, numerous donor–acceptor complexes have been generated

by covalently linking various redox-active inorganic complexes to surface amino-acid residues

of cyt c.58–60Interprotein electron transfer has also been examined using cyt c complexes withcytochrome c oxidase,61–65 plastocyanin,66–69 cyt b5,70–72 and cytochrome c peroxidase.73–76Sincecyt c is ubiquitous, easy to isolate, stable, and soluble, it has also become a system forthestudy of protein folding77–86 and protein dynamics.87–90

8.2.3.2 General Classifications

Cyts c fall into at least four general classes.5,91,92Thelargest class, class I, consists of small (8–

20 kDa), monoheme cyts c that arehomologous to mitochondrial cyt c Sequence and ral data have be e n use d to divide class I into sixte e n subclasse s.92 Mitochondrial cyts c andpurplebacterial cyts c2 makeup thelargest subclass Additional subclasses includePseudo-monascyts c551, cyts c4, cyts c5, cyts c6(algal cyts c553), Chlorobium cyt c555, Desulfovibrio cyts

structu-c553, cyanobacterial and algal cyts c550, Ectothiorhodospira cyts c551, flavocytochromes c,methanol dehydrogenase-associated cyt c550 or cL, cytochrome cd1 nitrite reductase, alco-hol dehydrogenase and its associated cytochrome subunit, Pseudomonas nitritereductase-associated cyt c, Bacillus cyt c, and Bacillus cytochromeoxidasesubunit II With thelargenumber

of cyts c presently being characterized structurally, proteins that fall into new additionalsubclasses continue to be found.93,94 Most class I cyts c arewater solubleand contain a6cLS heme Their heme-attachment site (–Cys–Xxx–Yyy–Cys–His–) is towards the N-terminus.The axial heme iron ligation is provided by the His residue of the heme-attachment site and aMet residue found near the C-terminus.Figure7(a) illustrates the general fold for class I cyts c.Class II cyts c havea singlec-type heme covalently linked to the highly conserved –Cys–Xxx–Yyy–Cys–His– sequence near their C-termini.5 While the number of conserved amino-acidresidues among class II cyts c is relatively small,5,95 the structural motif of a left-twisted, four helix bundle is characteristic of these proteins (Figure7(b)).96,97 A His residue occupies oneaxial coordination site of the heme iron, while the second axial coordination site is variable Thisclass has two subclasses that are distinguished by the spin state of the heme Subclass IIa consists

of cyts c0, which havehigh-spin (HS) configurations in thereduced form [FeII, S¼ 2] and either HS(S¼ 5/2)98–101 or a quantum-admixed spin (admixture of S¼ 5/2 and S ¼ 3/2) states98,100,102–104 inthe ferric form For example, the observed g-values for ferric Chromatium vinosum cyt c0(g1¼ 5.32, g2¼ 4.67, g3¼ 1.97) are not typical for a purely high-spin ferric heme, and simulation

of this EPR spectrum reveals that the electronic ground state of this cyt c0consists of 51% S¼ 3/2and 49% S¼ 5/2.100 These cyts c0 are found in photosynthetic and denitrifying bacteria.Subclass IIb includes proteins like cyt c556 from Rhodopseudomonas (Rps.)palustris,105 Rhodo-bacter (Rb.)sulfidophilus106 and Agrobacterium tumefaciens,5 and cyt c554 from Rb sphaeroides107that contain low-spin hemes In these cases, the sixth heme ligand, a Met, is found near theN-terminus

Triheme, tetraheme, octaheme, nonaheme, and 16-heme cyts c from sulfateand sulfur-reducingbacteria are included in class III.5,92,108–112Gene duplication of tri- and tetraheme units is clearlyapparent In Desulfovibrio species, the tetraheme proteins (15 kDa) are part of the electron-transfer chain that couples theoxidation of molecular hydrogen by hydrogenaseto sulfatereduc-tion.113 These multiheme proteins, also known as cyts c3, generally have bis-His heme ligation.Although the conserved amino acids in this class appear to be limited to the heme-binding cysteinesand the heme iron axial histidines, the three-dimensional structures of Desulfovibrio tetraheme cyts

c340,114–121

conserved.92 Interestingly, the heme–heme distances and heme–heme angles are evolutionarilyhighly conserved The hemes are in close proximity to one another, with adjacent pairs of hemeplanes nearly perpendicular to one another (Figure7(c)) There is, however, considerable variability

in the dihedral angle between the axial His planes in Desulfovibrio (D.) cyts c.119,120,122

Class IV consists of large (40 kDa) tetraheme photosynthetic reaction center (THRC) cyts c.Rsp viridis THRC cyt c is the electron donor to the bacteriochlorophyll special pair Thestructure of this membrane-associated Rsp viridis cyt c exemplifies the tertiary structure of thisclass (Figure7d).5 Its four hemes are in a linear arrangement, with alternating high- and low-potential sites While Rsp viridis THRC cyt c has three hemes with bis-His ligation and one withHis/Met ligation, the homologous THRC cyts c from Chloroflexus aurantiacus92and Rubrivivax(Rv.)gelatinosus123appear to contain only His/Met heme ligation.

Nitrosomonas (N.)europaeacyt c554is a tetraheme that is not homologous to either class III orclass IV multiheme cytochromes, and is considered to be in a class of its own (Figure7e).Involved in the biological nitrification pathway, this cyt c554accepts two electrons from hydro-xylamine oxidoreductase (HAO) upon generation of nitrite Its one high-spin heme and three6cLS hemes (þ47, þ47, 147, and 276 mV vs SHE) are arranged in two types of pairs wherethe hemes are in van der Waals contact Hemes III/IV have their porphyrin planes nearlyperpendicular to one another in an arrangement similar to that in cyts c3.6 Heme pairs I/IIIand II/IV have nearly parallel porphyrin rings that overlap at one edge, similar to the hemearrangement in HAO and cytochrome c nitrite reductase.6 Sequence similarities between theseseemingly unrelated proteins are found in the polypeptide near the hemes when the heme-stackingarrangement is used to align the protein chains.124Based on this and the conserved nature of theheme organization, it has been suggested that N europaea cyt c554, HAO, and cytochrome cnitrite reductase have a common evolutionary origin, but have diverged to fulfill differentfunctions

A sixth class of c-typecytochromes consists of thecyts f from thecyt b6fcomplex of oxygenicphotosynthesis The crystal structure cyt f on the lumin-side reveals two elongated domains made

up primarily of -sheet secondary structure, with the heme attached to the larger domain close to

(c) (c)

theinterfaceof thetwo domains (Figure7f).7 Its -sheet content and axial-heme ligation (His

of c-type cytochrome already discussed.7,125

8.2.3.3 Structural Studies of Mitochondrial Cytochromes c

As a protein subgroup of class I, mitochondrial cyts c have sequence homologies that areevolutionarily conserved.5,126 This sequence homology translates into a high degree of structuralsimilarity Crystal structures areavailablefor tuna,127–129horse,130rice,131and yeast (iso-1 and iso-2

of Saccharomyces (S.)cerevisiae)132–134mitochondrial cyts c Thetypical cyt c fold consists of a

a hydrophobic pocket Only five atoms at the edge of the heme pyrrole rings II and III are exposed

to the surface, while the heme propionates are shielded from bulk solvent by interactions withpolar side chains In some bacterial class I cyts the propionates are exposed to solvent, due tovariations in surfaceloops on thecommon class-I corestructure.5The features common to all fivemitochondrial cyt c structures include the heme attachment site at Cys14 and Cys17, His18 andMet80 as fifth and sixth heme iron ligands, hydrogen bonding from Pro30 to His18, hydrogenbonding from Tyr67 to Met80, heme propionate-7 hydrogen bonded by Gly41, Tyr48, and Trp59,and heme propionate-6 hydrogen bonded by Thr/Ser49, Thr78, Lys79 Cyt c from tuna, horse,and yeast (both isozymes) also has hydrogen bonds between Asn52 and propionate-7.135

The axial ligand bonds to Met80 and His18 are almost perpendicular to the pyrrole nitrogenplane of the heme The axial His orientation, as defined by an angle  (with respect to the Fe–NpIIbond vector inFigure1), is 46.5 in reduced iso-1-cyt c and 55.8 in oxidized iso-1-cyt c.133Thestereochemistry of the Met80 sulfur atom in eukaryotic cyts c, as demonstrated by X-ray crystal-lography and NMR NOE experiments, is generally in the R configuration.136

The heme is slightly saddled with pyrrole rings involved in the thioether bonds to the tide chain showing the greatest deviation from the mean porphyrin plane.133 This distortion isobserved in mitochrondial cyts c from various sources137 and when significant heme structuraldifferences are observed, they correlate with amino-acid variations in the –Cys–Xxx–Yyy–Cys–His– sequence.21 NSD results for wild-type iso-1-cyt c from yeast indicate that ruffling is themajor out-of-plane deformation Negative saddling and small positive amounts of waving deform-ations also contribute.20 Examination of 16 iso-1-cyt c mutants indicates that the relative con-tribution of each out-of-plane deformation to the total distortion is fairly constant Thus, thenature of heme conformational distortion appears relatively insensitive to alterations in the hemepocket or conserved amino-acid residues Shelnutt and co-workers have hypothesized that theheme distortion in cyts c is due to strain introduced through the covalent thioether linkages137bythe five residues, –Cys–Xxx–Yyy–Cys–His–,20,21,138 of the heme attachment sequence Recently,the iso-1-cyt c (Cys14Ser) variant with a single thioether bond between the heme and the proteinhas been shown to retain its Met80/His18 axial ligation.139 An NSD analysis of iso-1-cyt c(Cys14Ser) should be useful in evaluating the above hypotheses

polypep-8.2.3.4 Redox-linked Conformational Changes in Class I Cytochromes c

The first structural determinations of redox-dependent conformational changes were reportedfor tuna cyt c in 1980.127 Comparison of high-resolution structures currently available forreduced cyts c129,132,134 and oxidize d cyts c128,130,131,133 yields similar conclusions: namely,that theoverall protein fold of mitochrondial cyts c is not significantly altered upon oxidation.However, there are conformational changes localized in the heme pocket that have beenextensively discussed within the context of redox potential The largest consistently observed,redox-dependent changes occur in the vicinity of a conserved water molecule (Wat166) andinvolvethesidechains of Tyr67, Thr78, and Asn52 Thesesidechains arehighly conservedamong eukaryotic cyts c126 and participate in hydrogen-bonding networks involving the con-served water molecule, the Met80 heme ligand, and propionate-7

The redox-coupled movement of Wat166135is shown for S cerevisiae iso-1-cyt c inFigure8 Inreduced iso-1-cyt c, Wat166 is hydrogen-bonded to the side chain of Asn52, which is also linkedvia hydrogen bonding to propionate-7 In oxidized iso-1-cyt c, Wat166 moves 1.6 A˚ closer to theheme, and the hydrogen bond to Asn52 is lost Possible reorientation of the Wat166 dipole and itsmovement toward the heme iron in oxidized iso-1-cyt c suggest that it helps stabilize the positivechargeon thehemeiron Oxidation of iso-1-cyt c also results in weakening of the Trp59/propionate-7 hydrogen bonding, movement of the Asp60 side chain, and strengthening of pro-pionate-7 interactions with Gly41 and an internal water molecule, Wat121.133

The immediate environment of Met80 also exhibits redox-dependent changes The Met80sulfur-to-heme-iron bond length increases upon oxidation (2.35 A˚, reduced; 2.43 A˚, oxidized),while the His18 nitrogen-to-heme-iron bond changes by only 0.02 A˚ (1.99 A˚, reduced; 2.01 A˚,oxidized).133In the reduced form, the OH of Tyr67 hydrogen bonds to the sulfur of Met80 Loss

of this hydrogen bond upon oxidation stabilizes the positively charged heme by making Met80less electron-withdrawing and allowing Tyr67 to hydrogen bond with Wat166 Phe82 is withinvan der Waals contact of the Met80 side chain The Phe82 side chain is parallel to the plane of the

heme close to its solvent-exposed edge; its position with respect to the heme plane changes slightlyupon oxidation.133

The increased out-of-plane distortion of the ferric heme in the iso-1-cyt c132,133 suggests thatheme distortion could be correlated to change of oxidation state However, this is not observedfor tuna cyt c.127 Evaluation of the out-of-plane heme distortions in ten crystal structures ofmitochondrial ferro- and ferricyts c reveals no conserved redox-dependent differences in theruffling or x- and y-waving deformations.21

Examination of redox-dependent structural changes in solution by NMR spectroscopy isfacilitated by the availability of proton assignments for horse cyt c,140–142 tuna cyt c,143 andyeast iso-1-cyt c (Cys102Thr),144 a variant that does not dimerize Analysis of NMR pseudo-contact shifts suggests only subtle differences between oxidized and reduced cyt c.145–148Solutionstructures determined for both oxidation states of horse cyt c149–152and iso-1-cyt c153,154havethegeneral secondary structural features and overall protein fold found in the crystal structures TheNMR structures for horse cyt c show a difference in backbone atom positions (2.4 A˚ r.m.s.variation150; 1.4 A˚ r.m.s variation151,152) upon oxidation High-anglesolution X-ray scatteringexperiments155 detect comparable oxidation-dependent protein conformational changes Thesolution structures of horse cyt c indicate that the percentage of the solvent-exposed heme surfacearea increases from 7.53% to 14.6% upon oxidation.150 The dynamical features of horse andyeast cyt c, as probed by proton exchange and 15N heteronuclear relaxation NMR experiments,are responsive to the redox state of the heme.88,89,152–154 These data have been interpreted asincreased flexibility of the protein fold in the oxidized form

Pseudo-two-dimensional NOESY-TOCSY spectra149,156 and ePHOGSY-NOESY ments157 have been used to detect water molecules with long residence times in the structure.Although the general location of the conserved water (Wat166, iso-1; Wat1, horse) is similar to itsposition in the crystal structure, its movement upon oxidation appears to be 3.7 A˚ away from theheme iron,150,156rather than 1.6 A˚ toward the heme as in the crystal structure This translates intodifferences in the Wat166/Wat1-protein hydrogen-bonding network when comparing solution andcrystal structures of oxidized cyt c

experi-In a fashion similar to mitochondrial cyts c, heme propionate conformations are redox-linked

in someprokaryotic class I cyts c like Rb capsulatus cyt c2158–162and Monoraphidium brunii cyt

c6.163–165However, the role of a conserved water analogous to Wat166 in iso-1-cyt c is not clear.For example, the position of the conserved water molecule in Rsp palustris cyt c2is relativelyinsensitive to oxidation state.166In cyts c6from Chlamydomonas (C.)reinhardtii167and Scenedes-mus obliquus,168an internal water molecule is positioned between the two heme propionates Its

position is not equivalent to that of iso-1-cyt c Wat166, and it is insensitive to the heme oxidationstate Methylobacterium extorquens cyt cH, an electron donor to methanol oxidase in methyl-otropic bacteria, and Thermus thermophilus cyt c552, an electron donor to a ba3-typecytochrome

coxidase, are two examples of prokaryotic class I cyts c where the conserved water molecule, orhydrogen-bonding equivalent, is missing from their heme pockets.169–171 These observationssuggest that the usual hydrogen-bonding network involving the heme pocket water molecule isnot essential to electron transfer.171

8.2.3.5 Conformational Changes as a Function of pH in Class I Cytochromes c

Five states identified as a result of reversible, pH-induced, conformational transitions in oxidizedhorsecyt c172 aresummarized in Scheme 1 Since these states were first reported in 1941,considerable work has gone into their characterization At near-physiological pH, the 6cLSstate III is the predominant form and is considered to be the native protein conformation.Formation of state II can be monitored by the appearance of new absorbance bands at 530 nmand 625 nm, and the concomitant appearance of NMR features typical of an HS ferric heme.5,173Resonance Raman data also indicate the presence of a 6cHS ferric heme and an additional 6cLSferric heme for state II.174The 6cHS ferric heme is consistent with displacement of Met80 by awater molecule The 6cLS ferric heme is proposed to have less out-of-plane distortion and His18/His33 axial ligation At pH 0.3, a 5cHS species predominates (state I) Particular attention hasbeen paid to the conformational change between states III and IV, known as the alkalinetransition, which has a pKa of 8.5–9.5 depending on the species and solution conditions.5Resonance Raman evidence suggests that a similar conformational change occurs in cyt c when

it interacts with cytochrome c oxidase.64,65 The sulfur-to-iron charge-transfer band at 695 nmcharacteristic of Met–FeIII ligation is lost at alkaline pH, indicating that Met80 is no longerligated to the heme iron EPR and MCD data for alkaline horse ferricyt c indicatethat thehemeiron is still 6cLS.175Chemical shifts reported for alkaline horse ferricyt c areconsistent with lysine(Lys) coordination to theheme.176Both EPR175and 2D NMR studies176indicate the presence of

at least two alkaline species in horse ferricyt c above pH 9.5 Studies involving chemical fication of Lys residues did not yield unambiguous identification of the sixth axial ligand in alkalinehorseferricyt c.177

modi-Since the late 1980s, the ability to generate site-directed mutants of S cerevisiae iso-1-cyt c hasfacilitated characterization of transitions of state III! IV and stateIV ! V Studies of iso-1-cyt cand variants, (Lys73Ala), (Lys79Ala), (Lys86Ala), (Lys87Ala), and doublemutant (Lys73Ala/Lys79Ala), involving 1H-NMR, EPR, resonance Raman, and UV-visible spectroscopies, havedemonstrated that Lys73 and Lys79 replace Met80 as the sixth heme iron ligand in two con-formers of state IV.178–180The coordinated lysines in these two alkaline conformers are replaced,most likely, by a hydroxide ion provided by Wat166 to form two conformers with pKas above10(stateV).180Scheme 2illustrates the conformational equilibria of wild-type iso-1-cyt c between pH

7 and 12, as characterized by component analysis of their resonance Raman spectra.180

In wild-type iso-1-cyt c isolated from yeast, Lys72 is trimethylated; when this S cerevisiaeprotein is expressed in Escherichia (E.)coli, Lys72 is not trimethylated Although the iso-1-cyts cexpressed in E coli and in S cerevisiae have the same spectroscopic properties at neutral pH, the

pKafor thestateIII! IV transition is 0.6 pKaunits lower for the protein expressed in E coli.1

H NMR data for iso-1-cyt c and its (Lys72Ala) variant expressed in E coli have identified a thirdalkaline conformer, which has Lys72 coordinated to the heme.181 Sincecyts c from highereukaryotes are not trimethylated at Lys72, this suggests that Lys72 could be an axial ligand inalkaline horse and other higher eukaryotic ferricyts c.179,181

Kinetics of the alkaline transition have been characterized with pH-jump experiments.175,182–184The two-step mechanism in Scheme 3accounts for the pH dependence of the monoexponentialrate constants observed from neutral pH to pH 10.184The first step in the alkaline isomerization,deprotonation of a titratable group in the native protein, ‘‘triggers’’ the second step, which isheme iron ligand exchange This minimal kinetic mechanism does not consider five-coordinateintermediates or multiple alkaline conformers Biphasic behavior observed in pH-jump experi-ments above pH 10 suggests the formation of a transient HS heme intermediate in ferricyt c fromyeast183 and higher eukaryotes175 within the first 100 ms A species with similar spectroscopicfeatures is observed in the iso-1-cyt c (Phe82Trp) variant as a thermodynamically stable, HS formmaximized at pH 8.5 ResonanceRaman data for the(Phe82Trp) protein suggest that, in addition to

an obligatory 5cHS intermediate, there is a six-coordinate intermediate in which either Wat166 orTyr67 is coordinated to thehemebeforedisplacement by Met80 to form stateIII, or by Lys73 orLys79 to generatestateIV.183

Thereduction potential for alkalinecyt c is substantially lower than that for the native protein 1-cyt c, pH 10.4: Enative¼ þ 230 mV, Ealkaline¼ 230 mV; horsecyt c, pH 10.0: Enative¼ þ 255,

(iso-Ealkaline¼ 205 mV vs SHE).185

Although cyts c2exhibit a more positive reduction potential thantheir mitochondrial counterparts (Table1), the
E (EalkalineEnative), 407 mV to 440 mV, issimilar.186 This large reduction potential difference is attributed to the Met80 heme ligation instate III, and a proposed lower percentage of solvent-exposed heme in state III relative to state IV

energy between native and alkaline states of10 kcal mol1for iso-1-cyt c.185It has been proposedthat this constitutes a binary molecular switch.179,185

The identification of two state IV conformers suggests the existence of two such switchingmechanisms in iso-1-cyt c.179 The identity of the ‘‘trigger’’ group(s) whose protonation statedictates the conformation of the protein is presently unknown The titratable groups Lys73/Lys79,179 Tyr67,184,187 His18,188 heme propionate-7,189,190 and the conserved water in the hemepocket128have been suggested as possibilities The character of the pH-linked conformations isalso affected by the phylogenetically conserved Phe82 Phe82 replacement by Ser, Tyr, Gly, Ile, orLeu results in destabilization of ferricyt c by lowering the pKafor thealkalinetransition.182Thebasis of this effect will probably be revealed with identification of the trigger group

Thermal titration of cyts c from horse, cow, and tuna monitored by FTIR shows tional transitions akin to thealkalinetransition at 54C, 57C, and 50C, respectively.191Thereversible disappearance of the 695 nm band of state III in iso-1-cyt c upon moderate heatingcorrelates with the loss of the heme iron Met80 sulfur bond The rate constant for site exchangebetween iron-bound and free Met is 1.8 s1 These data, along with NMR data, suggest that the

N

Fe3+

OH?

NHHis18

high-temperature conformation at neutral pH and the alkaline conformation at room temperaturearethesamewith a Lys as with thesixth hemeligand.192 However, comparison of resonanceRaman spectra of Lys 73 and Lys 79 ligated alkaline states (IVa and IVb) with those of the high-temperature species do not support this conclusion.180,193,194

8.2.3.6 Intramolecular Heme Ligand Rearrangements

8.2.3.6.1 Mitochrondrial cytochrome c folding intermediates

Heme ligand switching as a mechanism for converting redox energy into ‘‘conformational’’ energyhas been observed in the alkaline transition (described inSection 8.2.3.5), in folding intermediateswith non-native heme ligands converting back to native Met/His coordination upon folding, and

in iso-1-cyt c (Phe82His) Although a detailed discussion of cyt c folding is beyond the scope ofthis chapter, the coordination chemistry of the heme plays an important role Apo-cyt c issubstantially unfolded in neutral solution Cyt c containing H2PPIX but no iron takes on afolded structure known as the ‘‘molten globule state,’’195which has a significant amount of native-like secondary structure, but its tertiary structure fluctuates Hence, maintenance of the rigidnativecyt c fold requires heme iron axial ligation Although the FeMet80 bond is not absolutelyrequired for native structure and function,196,197thedifficulty in obtaining cyt c His18 mutants198suggests that the FeHis18 bond is essential

Unfolded cyt c generated in urea or guanidine hydrochloride (GuHCl) contains a low-spin, His heme adduct In unfolded iso-1-cyt c, nativeaxial ligand His18 and oneof His26, 33, or 39ligatethehemeiron.199–201Upon folding, Met80 replaces the non-native His heme ligand Kineticfolding studies have suggested that slow-folding phases are due to non-native His heme liga-tion.201–204 This is substantiated by studies of cyts c that lack theproposed non-nativeligands.Folding kinetics for class I cyts c that contain only one His residue, like D vulgaris cyt c553and

bis-Rb capsulatus cyt c2, are not complicated by ligand-dependent kinetic phases.205,206 At neutral

pH cyt c553 folds within 220 ms,206 approximately a thousandfold faster than other cyts c undersimilar conditions.207–210 A dramatic increase in folding rate is also observed for the iso-2-cyt c(His33Asn/His39Lys) double mutant relative to wild-type protein.211 Equilibrium and stopped-flow kinetics with horse cyt c (His26Gln) and (His33Asn) mutants indicatethat His33 is thepredominant heme iron ligand in GuHCl-unfolded horse cyt c (pH 5.0).201,212

The heme ligation states observed under varying conditions for ferricyt c aresummarized in

fully denatured protein in neutral solution The species observed were dependent on the identity

of thedenaturant and its concentration.213,214In low concentrations of GuHCl, two intermediateswith Lys/His heme ligation were observed.214 Protein-folding kinetics and pH titration data fortuna cyt c, which lacks His33, suggest that Lys/His coordination also occurs in the misligated

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