Tài liệu Báo cáo khoa học: Open questions in ferredoxin-NADP+ reductase catalytic mechanism ppt

Measurements of electron transfer rates and binding equilibria indicate that NADPH and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fi

R E V I E W A R T I C L E

Ne´stor Carrillo and Eduardo A Ceccarelli

Molecular Biology Division, Instituto de Biologı´a Molecular y Celular de Rosario (IBR), Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Argentina

Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are

ubiquitous flavoenzymes that deliver NADPH or low

potential one-electron donors (ferredoxin, flavodoxin) to

redox-based metabolisms in plastids, mitochondria and

bacteria The plant-type reductase is also the basic prototype

for one of the major families of flavin-containing electron

transferases that display common functional and structural

properties Many aspects of FNR biochemistry have been

extensively characterized in recent years using a combination

of site-directed mutagenesis, steady-state and transient

kine-tic experiments, spectroscopy and X-ray crystallography

Despite these considerable advances, various key features in

the enzymology of these important reductases remain yet to

be explained in molecular terms This article reviews the

current status of these open questions Measurements of

electron transfer rates and binding equilibria indicate that

NADP(H) and ferredoxin interactions with FNR result in a

reciprocal decrease of affinity, and that this induced-fit step is

a mandatory requisite for catalytic turnover However, the

expected conformational movements are not apparent in the

reported atomic structures of these flavoenzymes in the free

state or in complex with their substrates The overall reaction

catalysed by FNR is freely reversible, but the pathways

leading to NADP+or ferredoxin reduction proceed through entirely different kinetic mechanisms Also, the reductases isolated from various sources undergo inactivating dena-turation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function

in vivo The mechanisms underlying this reductive inhibition are so far unknown Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency Using the formalism of the Albery–Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modifi-cation of their catalytic competence is relevant on both scientific and technical grounds

Keywords: ferredoxin-NADP(H) reductase; flavoproteins; oxidoreductases; ferredoxin; flavodoxin; catalytic mechanism; X-ray crystallography; steady-state kinetics; transient kinetics; enzyme evolution

Portrait of a reductase

Ferredoxin-NADP(H) reductases (FNR, EC 1.18.1.2)

con-stitute a family of hydrophilic, monomeric enzymes that

contain noncovalently bound FAD as a prosthetic group

The first FNR was isolated from pea thylakoids in the

mid-1950s [1] Shortly thereafter, Shin and Arnon [2] showed

that the physiological role of the chloroplast reductase was

to catalyse the final step of photosynthetic electron

trans-port, namely, the electron transfer from the iron-sulphur

protein ferredoxin (Fd), reduced by photosystem I, to NADP+ (Eqn 1) This reaction provides the NADPH necessary for CO2assimilation in plants and cyanobacteria

2 FdðFe2þÞ þ NADPþþ Hþ ƒƒƒ!ƒƒƒ 2 Fd ðFe3þÞ

þ NADPH ð1Þ Equation 1 reflects one of the most conspicuous features of FNR, its ability to split electrons between obligatory one-and two-electron carriers, which is a direct consequence of the biochemical properties of its prosthetic group FAD and other flavins (Fl) can exist in three different redox states: oxidized, one-electron reduced (semiquinone) radical and fully reduced hydroquinone, containing, respectively, 18, 19 and 20 electrons in a p orbital system constructed from 16p atomic orbitals [3] The isoalloxazine ring also has 7r lone electron pairs available for protonation, providing ample opportunity for tautomers

When free in solution, flavin semiquinone radicals disproportionate to the oxidized and reduced forms (Eqn 2), but when buried in a protein they are much less prone to dismutation This results in a considerable stabilization of the semiquinone, which in turn allows flavoproteins, in general, to readily engage in mono- and

Correspondence to N Carrillo, IBR, Facultad de Ciencias

Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario,

Suipacha 531 (S2002LRK) Rosario, Argentina.

Fax: + 54 341 439 0465, Tel.: + 54 341 435 0661,

E-mail: carrill@arnet.com.ar

Abbreviations: cyt c, cytochrome c; Fl, flavin; Fd, ferredoxin;

FNR, ferredoxin-NADP(H) reductase; Fld, flavodoxin;

ox, oxidized; red, reduced; sq, semiquinone.

Enzyme: Ferredoxin-NADP(H) reductases (FNR, EC 1.18.1.2).

(Received 13 January 2003, revised 4 March 2003,

accepted 13 March 2003)

bielectronic exchange reactions This disposition is favoured

in FNR by the small differences existing in the redox

potentials (Em) for the various one- and two-electron

transfer processes, that is )331, )314 and )323 mV for

the oxidized/semiquinone couple, the

semiquinone/hydro-quinone pair and the two-electron FAD reduction,

respect-ively

H2FlÆþ H2FlÆ ƒƒƒ!ƒƒƒ HFloxþ H2Flredþ Hþ ð2Þ

FNR functions are by no means confined to

photosyn-thesis Following the isolation of the chloroplast reductase,

flavoproteins with FNR activity were found in

photo-trophic and heterophoto-trophic bacteria, animal and yeast

mitochondria, and apicoplasts of obligate intracellular

parasites [4–10] Studies in a number of species showed

that FNR operates as a general electronic switch at the

bifurcation steps of many different electron transfer

pathways In heterotrophic organisms and tissues,

inclu-ding roots and other nonphotosynthetic plant organs, the

reaction represented by Eqn (1) is driven toward Fd

reduction, mobilizing low-potential electrons for a diversity

of metabolisms They include steroid hydroxylation, nitrate

reduction, nitrogen and hydrogen fixation, anaerobic

pyruvate assimilation, desaturation of fatty acids and

synthesis of amino acids and deoxyribonucleotides (Table 1,

reviewed in [11]) Van Thor et al [12] have proposed that

this
backward reaction might also occur in photosynthetic

cells of cyanobacteria, being a rate-limiting step of cyclic

electron flow around photosystem I

Some bacteria and algae possess another flavoprotein,

FMN-containing flavodoxin (Fld), that is able to

effi-ciently replace Fd as the electron partner of FNR in

different metabolic routes, including photosynthesis [8]

In cyanobacteria, Fld expression is induced under

condi-tions of iron deficit, when the [2Fe)2S] cluster of Fd

cannot be assembled [8] In other prokaryotes, flavodoxins

are synthesized constitutively, or induced by oxidants

[13] Both Fld and FNR participate in detoxification of

reactive oxygen species in aerobic and facultative bacteria

[13,14]

FNR displays a strong preference for NADP(H) and is a

very poor NAD(H) oxidoreductase In contrast, the site of

electron donation from reduced flavin appears to be open to

a remarkable variety of adventitious oxidants of very

different structure and properties The reaction is largely

irreversible and led the original FNR discoverers to label it

as a thylakoid-bound
NADPH diaphorase [1]

NADPHþ Hþþ nAox! NADPþþ nAred ð3Þ

Aoxand Aredrepresent the oxidized and reduced forms of the electron partner, and the term n equals one or two depending on whether the oxidant behaves as a two- or a one-electron carrier, respectively The list of acceptors comprises ferricyanide and other complexed transition metals, substituted phenols, nitroderivatives, tetrazolium salts, NAD+ (transhydrogenase activity), viologens, qui-nones and cytochromes (reviewed in [15]) By contrast, the oxidase activity of FNR is very low [15,16], suggesting that there might be restrictions to the formation of the caged radical pair and/or the covalent (C4a)-flavin hydroperoxide intermediates required for efficient oxygen reduction [17], although other mechanisms cannot be ruled out The oxidase reaction is enhanced several-fold by many FNR acceptors, including one-electron reduced Fd or Fld, viologens, nitroderivatives and quinones, that can readily engage in oxygen-dependent redox cycling leading to superoxide formation [16,18] Diaphorase activity is prob-ably devoid of physiological meaning in most cases, but it has paid an enormous service to the understanding of FNR function and catalytic mechanism In addition, some of these artificial reactions might have technological relevance for bioremediation and the pharmaceutical industry [18,19] This brief account illustrates the plasticity of FNR as a catalyst and its ubiquity among living organisms Studies on the enzymology of this reductase began in the late 1960s, employing both steady-state and rapid kinetic measure-ments, and a comprehensive model describing the various steps of reaction 1 was formulated by Batie and Kamin in

1984 Since then, an impressive amount of information on FNR structure, function and biogenesis has been gathered, providing clues to understand holoenzyme assembly, sub-strate binding and catalysis in molecular terms However, several key features of FNR enzymology remain obscure, and could not be accounted for, or reconciled with the available structural-functional data The aim of this article is

to review these open questions in FNR biochemistry, as a tool and conceptual framework for future research It is worth noting that a thorough understanding of FNR catalytic mechanism has become increasingly important after the recognition of this reductase as the prototype for a

Table 1 Functions associated with ferredoxin-NADP(H)reductase in different organisms.

Fatty acid desaturation

large family of flavin-containing enzymes displaying similar

structures and reaction mechanisms [20–26]

The two-domain structure of

ferredoxin-NADP(H) reductases

Three-dimensional models of the oxidized and fully reduced

forms of spinach FNR, refined to 1.7 A˚ resolution, were

first reported by Karplus and coworkers [20–23] The

flavoprotein molecule is made up of two structural domains,

each containing approximately 150 amino acids (Fig 1A)

The carboxyl terminal region includes most of the residues

involved in NADP(H) binding, whereas the large cleft

between the two domains accommodates the FAD group

The isoalloxazine ring system is tightly bound to the amino

terminal domain through hydrogen bonds and van der

Waals contacts [23] It stacks between the aromatic side

chains of two tyrosine residues, represented in spinach FNR

by Tyr96 on the si-face and Tyr314 (the carboxyl terminus)

on the re-face (Fig 1B) The phenol ring of Tyr314 and the

flavin group are coplanar in such a way as to maximize

p-orbital overlap [20] A large portion of the isoalloxazine

moiety is shielded from the bulk solution by the side chain

of the carboxyl terminal tyrosine, but the edge of the

dimethyl benzyl ring that participates in electron transfer

remains exposed to solvent in the native holoenzyme [23]

Crystal structures have also been resolved for other FNR

proteins, including those present in pea, paprika and maize

chloroplasts [27–29], corn root plastids [30], cyanobacteria

[31], Escherichia coli [32] and Azotobacter vinelandii [33]

Despite ample variations in amino acid sequences, the chain

topologies of all these proteins are highly conserved, with

most differences occurring at the loops between the

invariant secondary structure elements [20–23,27–33] In

the chloroplastic and cyanobacterial reductases, as in most

flavoproteins, the FAD molecule binds in an extended form,

with the 2¢-P-AMP moiety wrapped up by a short

sheet-loop-sheet motif of the apoprotein [20–23,27–31] In

contrast, the adenosine in the E coli FAD bends back

from the diphosphate so that the nitrogen at position 7 of

the adenine group forms a hydrogen bond to nitrogen 1 of

the isoalloxazine This interaction is further stabilized by

stacking of the two terminal aromatic side chains; the

phenol ring of Tyr247 with the flavin, as in plant FNR, and

the indole of Trp248 with the adenine [32]

The FAD cofactor of the A vinelandii FNR is also twisted

and stabilized in a similar way, but the environment of the

prosthetic group presents some unique features in this

reductase The most important difference is the absence of

the aromatic interaction on the re-face of the isoalloxazine

that is typical of plant, cyanobacterial and E coli FNR

proteins [33] Instead, the tyrosine position in the Azotobacter

enzyme is occupied by an alanine (Ala254) Despite these

major modifications in the active site region, the FMN halves

of FAD display very similar conformations when the various

homologous proteins are superimposed [20–23,27–33]

Several amino acid residues important for the structural

integrity of the native holoenzyme, for electron transfer, and

for FAD, NADP(H), ferredoxin and flavodoxin binding,

have been identified using a combination of chemical

modification experiments, site-directed mutagenesis and

X-ray diffraction studies This very interesting aspect of

FNR biochemistry will only be addressed here in relation to the catalytic mechanism of the enzyme Further information

on these topics can be found in a number of articles and reviews [23,31,34–57]

Finally, it is noteworthy that not all flavoenzymes displaying FNR activity belong to the FNR class The adrenodoxin reductases found in animal and yeast mito-chondria and their bacterial homologues represent a curious case They are hydrophilic, monomeric proteins made up of FAD and NADP(H) domains that can freely exchange electron partners (ferredoxin, flavodoxin, adrenodoxin) with plant-type FNR [58] Many features of NADP(H) docking and catalysis are also similar, although reaction geometry is different [58] However, mitochondrial reduc-tases are unrelated in sequence to their chloroplast coun-terparts, and the structural data indicate that they actually belong to the disulphide oxidoreductase family of flavopro-teins, whose prototype is glutathione reductase [6,58] The plant-type and mitochondrial-type FNR progenies thus represent two different and independent origins, followed by

a remarkable case of convergent evolution to yield proteins with essentially the same enzymatic properties

The reaction pathway

Batie and Kamin [59,60] formulated the first detailed pathway for the FNR-mediated electron transfer between NADP(H) and ferredoxin, using data from binding equili-bria, steady-state kinetics and rapid mixing experiments (Fig 2) The overall reaction was interpreted as an ordered two-substrate process, with NADP+binding first Under these assumptions, the kinetics were shown to be consistent with the formation of ternary complexes as intermediates of the catalytic mechanism (Fig 2, steps 2 and 5) Substrate-binding parameters and rate constants were determined for the complete pathway mediated by both plant and bacterial reductases, and for several individual steps (Table 2) Turn-over numbers in the range of 200–600 s)1have been reported for the spinach and Anabaena enzymes, whereas E coli FNR is much less active (Tab le 2) Go´mez-Moreno and coworkers have also studied the electron transfer to and from flavodoxin [46,47 and references therein] The reverse reaction, that is the electron transport from NADPH to Fd (or Fld), is routinely measured in vitro through a coupled assay, using cytochrome c (cyt c) as final electron acceptor (Eqns 4 and 5)

NADPHþ 2 Fd ðFe3þÞ ƒƒƒ!ƒƒƒ NADPþ þ Hþ

þ 2 Fd ðFe2þÞ ð4Þ

FdðFe2þÞ þ cyt cðFe3þÞ ! FdðFe3þÞ þ cyt cðFe2þÞ ð5Þ

In the following sections, we will analyse the various steps involved in FNR catalysis, with emphasis in unresolved or controversial questions Most of the discussion will be based

on data obtained with the plant and cyanobacterial flavoenzymes The reductases present in E coli and other bacterial species are less well characterized, but it is already clear that they display a number of differences with respect

to their plant-type counterparts These distinct features will also be addressed when pertinent

Fig 1 The Ca polypeptide backbone and the active site region of plant-type ferredoxin–NADP(H)reductase (A) FNR is a two-domain flavoprotein The computer graphic is based on X-ray diffraction data for the spinach enzyme [23], with the FAD binding domain shown in blue, the NADP(H) binding domain in pink, and the FAD prosthetic group in yellow (B) Detailed view of the isoalloxazine ring system in FNR, displaying relevant interactions with active site amino acid residues The phenol ring of the carboxyl terminal tyrosine 314 shields the re-side of the flavin from solvent The figure was drawn using - 3.7 and rendered with -

NADP(H) binding (Fig 2, steps 1 and 9)

Even though no chemistry is expected to occur between the

oxidized nicotinamide and the oxidized flavin, the role of

NADP+ as leading substrate during FNR turnover is

supported by a number of kinetic measurements In the

spinach enzyme, electron transfer from reduced Fd to FNR

is too slow (kobs¼ 40–80 s)1) to account for steady-state

rates of nucleotide reduction (kobs> 500 s)1) NADP+

binding greatly accelerates this reaction (Table 2), indicating

that the presence of the coenzyme at the active site is a

prerequisite for catalysis [60] Possible mechanisms involved

in this activation are discussed in the next section

Formation of the binary complex has been measured

in vitro by a variety of techniques, most conspicuously

differential spectroscopy Binding isotherms fit to simple

hyperbolic functions in all cases Dissociation constants

increased with the ionic strength of the medium (I) and the

Fd concentration, with K ¼ 6–20 lMat I 100 mMfor

plant and Anabaena reductases [43,44,50,59,61] Karplus and coworkers [20,23] have proposed that stacking of the nicotinamide ring onto the re-face of the isoalloxazine moiety during enzyme turnover requires displacement of the aromatic side chain of the carboxyl terminal tyrosine This thermodynamically unfavoured process results in a decrease

of the binding affinity for NADP(H) relative to those of the 2¢-P-AMP and 2¢-P-ADP-ribose analogues [61] A similar movement has been postulated for the penultimate tyrosine residue of E coli FNR, although in this case the carboxyl terminal tryptophan interacting with the adenosine moiety

of FAD might also undergo a conformational change upon substrate binding [32,62–64]

Complex formation is then interpreted as a two-step binding of the nucleotide to a bipartite site (Fig 3) The first step involves a strong interaction of FNR with the adenosine part of NADP(H), followed by isomerization leading to nicotinamide docking and, eventually, hydride transfer (Eqn 6)

FNRþ NADPðHރƒƒ!ƒƒƒ FNR NADPðHÞ

ƒƒƒ!ƒƒƒ FNR NADPðHÞ ð6Þ Where FNRÆNADP(H) and FNRÆNADP(H) represent complexes with the coenzyme bound through the adeno-sine, or the adenosine and nicotinamide portions, respect-ively The second step in Eqn 6 is energetically costly and weakens the entire interaction to a remarkable extent Measurements of complex formation in vitro led to the amazing conclusion that under saturating conditions less than 20% of the nicotinamide is placed in contact with the flavin, and is therefore available for hydride transfer [50] The model of Fig 3 explains why crystals of FNR with bound analogues could be obtained with the wild-type enzyme, whereas those involving NADP(H) complexes were only possible with engineered FNR proteins in which the carboxyl terminal tyrosine had been replaced by nonaromatic residues such as serine [27] In these FNR mutants, rearrangement of the phenol group is no longer required, and both pockets of the binding site would be readily accessible

Figure 4 shows amino acid residues involved in recogni-tion of the adenosine-ribose and the NMN porrecogni-tions of the dinucleotide, as identified through structural and mutage-nesis studies [23,27,31,51] They include residues displaying charge interactions with the specific 2¢-phosphate group presumably responsible for discrimination against NAD(H) The increase in coenzyme affinity caused by substitutions of the carboxyl terminal tyrosine was so dramatic that the resulting FNR mutants were able to avidly incorporate NADP+during biosynthesis in E coli and keep it bound through the purification and crystallization steps [27] Incidentally, once the carboxyl terminal residue is replaced, the electrostatic interactions at the 2¢-phosphate group of NADP+are no longer sufficient to discriminate between the coenzymes, and FNR becomes an efficient NAD(H) oxidoreductase [50]

The reversible NADPH release depicted in step 9 also represents the initial event of the reverse reaction, NADPH-ferredoxin reductase, and will be addressed in further detail

in a forthcoming chapter, when discussing that FNR activity

Fig 2 The electron transfer mechanism of ferredoxin-NADP(H)

reductase The various steps of the catalytic pathway were initially

proposed by Batie and Kamin [60] on the basis of kinetic and binding

experiments on the spinach FNR Oxidized forms are white,

one-electron reduced forms are light grey and two-one-electron reduced forms

are dark grey.

Electron transfer from reduced ferredoxin

to ferredoxin-NADP(H) reductase

(Fig 2, steps 2–4)

A recent article by Hurley et al [57] has provided a very

comprehensive and updated review of experiments

descri-bing the interaction and electron transfer between Anabaena

FNR and Fd Accordingly, the present section gives only a concise account of these data; the reader is referred to the above mentioned article for a more detailed description of the two processes

Conversion of oxidized FNR to the semiquinone form (FNRsq) by reduced Fd (or Fld) is too fast to be measured

by rapid mixing techniques [44,60,65] However, the kinetics

Fig 3 Schematic representation of the bipartite NADP(H)-binding mode to ferredoxin–NADP(H) reductase The model is based on the properties of

a tyrosine-to-serine site-directed mutant of pea FNR bound to NADP(H) [27,50] Sites A and N represent the adenosine- and the nicotinamide-binding regions, respectively, in the active site of FNR.

Table 2 Kinetic and binding parameters for various activities and interactions of ferredoxin-NADP(H)reductase Binding and kinetic parameters were averaged from experiments carried out at I £ 100 m M (Original sources cited in the text.) When dispersion among reported values exceeded 50%, the interval between extreme data is indicated ND, not determined.

Reaction

FNR source

K m or K d (l M ) k obs or k cat (s)1) Spinach leaves Anabaena E coli Spinach leaves Anabaena E coli Binding

Electron transfer

Fd red fi FNR fi NADP +

1b

60c

6200 (>600) c,a

250 (>600)c,a

8 (25) a

ND (8)a FNR red fi NADP +

33 b

4b

7 b

200d

>600 d

>140d

22 FNR red fi Fd ox (Fld ox ) ND ND <5 (<5) a ND >600 (3) a 2 (0.01) a

NADPH fi FNR fi K 3 Fe(CN) 6 30 b

100b

23 b

170b

10 b

24b

a Values in parentheses are those obtained with Fld b The upper and lower numerals indicate K m estimates for electron donors (Fd red , NADPH) and acceptors (NADP + , Fd ox , Fld ox , K 3 Fe(CN) 6 ), respectively c The upper and lower values reported provide rates of transfer for the first and second electron, respectively.dThe upper and lower numerals show rates of formation of charge–transfer complexes and rates of hydride transfer, respectively.

of this reaction could be resolved for the Anabaena

reductase by using laser flash photolysis, yielding a kobsof

about 6000 s)1and a Kdof 1.7 lMfor the transient FNRox–

Fdred complex at I¼ 100 mM [40,44,57] Taking into

account the many contacts that are required to establish

this protein–protein interaction, it is somehow surprising

that FNR can efficiently accommodate Fd or Fld, two

proteins that differ completely in their primary, secondary

and tertiary structures Other similarities must exist to

account for their functional equivalence, in spite of the lack

of homology It is interesting to note, in this context, that

the molecular association of FNR with its electron partners

is steered by electrostatic interactions [37,40,47,56,66,67]

Using the Hodgkin index as a similarity measure, Ullmann

et al [68] showed that Fd and Fld could be completely

overlapped on the basis of their surface electrostatic

potentials The active sites and prosthetic groups of both

proteins, rather than their centres of mass, coincided in the

alignment [68]

Transient associations of the electron carriers in different

oxidation states are generally not amenable to structural

studies, but binary complexes of oxidized FNR and Fd

could be resolved by X-ray crystallography for both the

Anabaenaand maize couples [29,69] The resulting

struc-tures provided insightful data to complement chemical

cross-linking and mutagenesis studies, and helped to model

flavodoxin docking [46] Fd binds to a concave region of the

FAD domain of maize FNR (Fig 5A), burying an accessible area of 800 A˚2 in each partner, which repre-sents about 5% and 15% of the total surface areas of FNR and Fd, respectively [29] The FAD and [2Fe)2S] redox centres are sufficiently close (6.0 A˚) for direct electron transfer through the space between the two prosthetic groups (Fig 5A–C) Distribution of surface charge and calculations of the molecular dipole moments confirm the relevance of complementary patches of basic and acidic residues in FNR and Fd, respectively [66,67] These polar groups play a major role in determining the relative orientation of the two electron carriers in the initial nonproductive complex [40,66] Attainment of the func-tional conformations competent for electron exchange requires further, fine adjustments, stabilized by a combina-tion of well-defined hydrogen bonds, salt bridges, van der Waals interactions and hydrophobic packing forces origin-ating from the dehydration of the protein–protein interface [40,47,48,52,57] When the FNR molecules of the corn and cyanobacterial complexes are superimposed, the Fd part-ners appear rotated by an angle of 96, indicating that many protein–protein interactions are different in the two systems [29,69] Hurley et al [57] have therefore proposed that, as in the case of Fld binding, the crucial parameters selected during evolution might be proximity of the prosthetic groups in a nonpolar environment to facilitate direct electron transfer

Fig 4 The nucleotide-binding site of ferredoxin–NADP(H)reductase View of NADP(H) bound to the pea FNR-Y308S mutant reveals the intimate interactions made by both the 2¢-P-AMP portion of the ligand and the nicotinamide NADP(H) is depicted in blue, FAD in yellow, amino acids in grey Dashed lines mark interactions of £ 3.5 A˚ that may engage in hydrogen b onds Residues are lab elled as in pea FNR (numb ers in parentheses are those of spinach FNR) The figure shows two water molecules that display hydrogen interactions with both NADP(H) and the protein backbone Modified from Deng et al [27], drawn using SWISS - PDBVIEWER 3.7 and rendered with POV - RAY

Full reduction of ferredoxin-NADP(H)

reductase (Fig 2, steps 5–7)

When spinach FNRox is mixed with excess Fdred in a

stopped flow system, all the flavoprotein is converted into

the semiquinone form in the dead time of the instrument

[60] Transfer of the second electron, however, is too slow to

be compatible with steady-state catalysis, as already indica-ted The latter process actually involves various steps, which are the dissociation of Fdox(Fig 2, step 4), binding of Fdred (step 5) and flavin reduction (step 6) The reaction is strongly inhibited by Fd and stimulated by NADP+,

Fig 5 Structure of the ferredoxin–NADP(H)reductase–ferredoxin complex (A) View of the maize leaf bipartite FNR–Fd complex with the ribbon diagram of Fd coloured in red, the FAD-binding domain of FNR in blue and the NADP(H) binding domain in pink (B) Hypothetical tripartite NADP(H)–FNR–Fd complex View of the superposition of the maize leaf bipartite FNR–Fd complex and the pea FNR–NADP(H) complex Pea and maize FNR polypeptides were superimposed by the least square fitting of the isoalloxazine ring of FAD The two domains of maize FNR are shown in light blue, Fd in red, FAD in yellow and the NADP(H) from the pea FNR–NADP(H) crystals in deep blue White arrows indicate the [2Fe )2S] cluster (green), the isoalloxazine ring of FAD and the nicotinamide ring of NADP(H) (C) Superimposed view of the active site structure

of the maize FNR complexed with Fd (in red), the free maize FNR (in light blue) and the pea FNR complexed with NADP(H) (in grey) The pyridine nucleotide is represented in blue Note that in the three superimposed structures E312 (306) undergoes significant movements upon complex formation To facilitate the observation, E306 from pea FNR was omitted in the main figure and included in the inset The models were based on the detailed structures of the bipartite complexes reported by Kurisu et al [29] and Deng et al [27] The figure was drawn using SWISS

-PDBVIEWER 3.7 and rendered with POV - RAY

indicating that step 4 is the rate-limiting step, and that

NADP+facilitates Fdoxrelease, allowing the entire reaction

to proceed at a rapid pace through steps 4 and 8 [60] The

sequence of events depicted in Fig 2 agrees well with other

experimental observations A ternary complex between the

two oxidized proteins and NADP+ is readily formed

in vitro as measured by differential spectroscopy The

affinity of FNR for Fdoxdecreased > 10-fold on addition

of NADP+, and vice versa, indicating a strong case of

negative cooperativity for binding [59] Essentially the same

results were obtained when using NADPH [59] In this case,

dissociation of the two enzyme–product complexes, rather

than formation of the FNR–substrate species, are the most

demanding steps that limit turnover

NADP+reduction and product release

(Fig 2, steps 8 and 9)

Full FNR reduction renders a two-electron reduced Fdox–

FNRred–NADP+complex that requires hydride transfer to

the nucleotide and dissociation to complete the catalytic

cycle The sequence of steps proposed in Fig 2 complies

with a canonical compulsory order mechanism, although

alternative pathways could be envisaged For instance, it is

conceivable that Fdoxcould dissociate from the FNRred–

NADP+ complex prior to nucleotide reduction Rapid

mixing experiments provided the main empirical support for

the proposed reaction order, showing that in a mixture of

the three components, the oxidation of FNRredb y NADP+

was faster than electron transfer from Fdredto the reductase

[60] Even in the absence of Fd, NADP+ reduction by

FNRred proceeds at  500 s)1, a rate compatible with

catalysis [42,60]

Open question: the molecular bases of

cooperativity

As indicated previously, interactions of ferredoxin and

NADP+with FNR exhibit reciprocal negative

cooperati-vity, which is translated, paradoxically, into positive

cooper-ativity at the kinetic level [60,61] It was expected therefore

that complex formation should lead to modifications in the

structure of the active site of the flavoprotein

Conforma-tional movements resulting from nucleotide binding,

how-ever, appear to be largely restricted to displacement of the

carboxyl terminal tyrosine, as judged by the crystal structures

of wild-type and mutant FNR in complex with NADP(H)

and analogues [23,27,31] We speculate that motion of the

phenol ring of tyrosine is responsible for the decrease in FNR

affinity for Fd, but the actual position of this residue when

pushed away by the entering nicotinamide is unknown In a

tyrosine-to-tryptophan mutant of pea FNR that allows for

 40% of nicotinamide occupancy, the displaced indole ring

failed to adopt a single ordered position [27]

Interaction of FNR and Fd, on the contrary, does lead

to structural changes in the two electron carriers relative to

the conformations of the free proteins [29] On complex

formation, the NADP(H) domain is displaced slightly as a

single unit, and the side chain of Glu312 (numbering of

spinach FNR) moves to hydrogen bonding distance of the

hydroxyl group of Ser96 [29] These two residues are highly

conserved among reductases of different origins, and their

charge, size and polarity are crucial to optimize the active site geometry for electron and hydride transfer [38,43, 45,49] The protein–protein interaction also affects the microenvironments of the two prosthetic groups The redox potentials (Em) of Fd and FNR were shifted by)25 mV and +20 mV, respectively, facilitating electron transfer in the photosynthetic direction, namely, from Fdredto FNRox [70]

It is not clear how these observed or putative structural changes in the active site region of FNR correlate with the induced-fit mechanism deduced from kinetic measurements Dorowski et al [28] have proposed that Fd binding might favour displacement of the carboxyl terminal tyrosine by nestling the phenol group into a hydrophobic pocket of the iron–sulphur protein These authors advanced further and challenged the model of Fig 2 by suggesting that Fd is the leader substrate that assists in NADP+binding Although such a mechanism would be at odds with the reported decrease in NADP+affinity upon Fd attachment, the two models can be reconciled by assuming that the dinucleotide binds first in a nonproductive manner through its 2¢-P-AMP portion Fd may then interact with the tyrosyl residue, favouring nicotinamide docking and establishing a loosely bound complex compatible with turnover [28] It is clear that the carboxyl terminal tyrosine plays a pivotal role during FNR catalysis, but further research will be required

to understand its actual function and importance

The backward reaction is a mechanistic puzzle

Ferredoxin (or flavodoxin) reduction is the most widely distributed function of FNR-type proteins Table 1 pro-vides a summary of metabolic routes that require such an activity from either FNR or adrenodoxin reductase In cyanobacteria, a single FNR species functions as NADP+ reductase in vegetative cells and as Fd reductase in heterocysts [8], whereas two distinct isoforms fulfil these roles in chloroplasts and nonphotosynthetic plastids of vascular plants In the latter case, tissue specificity is determined at the transcriptional level by cis-acting regula-tory elements [71,72] Interestingly, the redox potentials of these reductases and those of their corresponding ferredox-ins have been tuned by evolution to favour the physiological direction of electron transport [30] However, the four proteins can be readily exchanged in vitro when assayed in a variety of reactions, indicating that the major force driving NADP+or Fd reduction in vivo would be the availability of substrates [30,73]

NADPH binding to oxidized FNR leads to rapid hydride exchange between the nucleotide and the reductase, result-ing in a succession of charge-transfer complexes involvresult-ing flavin and nicotinamide (Eqn 7, species in brackets), whose formation can be followed by the appearance of long wavelength absorbance signals [61]

FNRoxþ NADPH ƒƒƒ!ƒƒƒ FNR½ ox NADPH

ƒƒƒ!ƒƒƒ FNR redNADPþ

ð7Þ The presence of various molecular species complicates the quantitative estimation of binding equilibria Batie and Kamin [61] obtained an upper limit of about 2 lMfor the

Kdof the spinach FNRoxÆNADPH complex, indicating that

nucleotide binding to FNRoxis tighter in the reduced state.

Complex formation is very rapid (kobs> 500 s)1), followed

by slower hydride transfer to the flavin at 200 s)1(Table 2)

Electron transfer from FNRred to Fdoxis too fast to b e

followed by stopped-flow techniques [43,44,52] All the

previous reactions proceed at velocities that are compatible

with the steady-state rate of Fd reduction (kobs¼ 200–

250 s)1), as measured by the cyt c reductase assay

[30,34,35,43,44,52,57]

The reversible nature of the various steps involved in

FNR-mediated NADP+reduction suggested that electron

transfer from NADPH to Fd should proceed by a reversed

version of the ordered pathway of Fig 2 The forward and

backward reactions are shown, in Cleland’s notation, in

Fig 6A,B, respectively Assuming that v¼ k)6[FNRox]

[NADPH]) k6[FNRoxÆNADPH + FNRredÆNADP+], then

in the absence of products the velocity equation for Fd

reduction (Fig 6B) will be:

Where Km, Kdand Vmhave their conventional meanings

and Km¢(Fd) represents the sum of the Kmvalues for the

successive interactions of the two molecules of Fdoxwith

FNRred and FNRsq (Fig 6B) Equation 8 predicts that

double reciprocal plots of v against the concentrations of any

of the two substrates should yield straight lines intersecting

in the fourth quadrant, as it occurs with the forward

reaction There is no simple way to measure Fd (or Fld)

reduction, because the reduced acceptor is reoxidized by

dissolved oxygen with kcat 40 s)1[16] Therefore, kinetic

evaluation of the Fd reductase activity requires

measure-ments under strict anaerobiosis, and experimeasure-ments of this kind

have not yet been carried out with the plant-type

flavopro-teins As indicated before, cytochrome c is usually employed

as a final acceptor that competes favourably with dioxygen for the spare electron of reduced Fd (Eqns 4 and 5) In vivo, the physiological acceptor enzymes (such as thioredoxin reductase, nitrate reductase or dihydroascorbate reductase) would play a similar role, preventing accumulation of the reduced
doxins that, under the oxygen tensions prevailing

in aerobic cells, could otherwise facilitate the propagation of superoxide radicals and other toxic oxygen derivatives Wan and Jarrett [63] have measured the anaerobic oxidation of NADPH by an E coli system made up of FNR and either ferredoxin or any of the two bacterial flavodoxins This reductase species is remarkably slow, turning over at 0.15 s)1for Fd and about 0.004 s)1for the flavodoxins [63] The rates of individual electron transfer steps are consistent with this slow pace of catalysis ([62,63], summarized in Table 2) Moreover, E coli FNR mediates direct reduction of cytochrome c at 5 s)1, with this rate being enhanced only about twofold by the addition of

saturating flavodoxin [62] Similar low activities have been obtained with the A vinelandii [74] and Rhodobacter capsulatus (C Bittel, N Carrillo and N Cortez, IBR, Rosario, Argentina, unpublished observations) reductases The collected results indicate that these bacterial FNR forms display distinct catalytic features, and their comparison with the plant-type enzymes needs to be considered with caution Surprisingly, when spinach Fd reduction was measured

in vitroby the cyt c assay, parallel lines were obtained in 1/v

vs 1/[NADPH] plots, suggesting a two-step transfer
ping-pong mechanism without formation of a ternary complex [75] The diaphorase activity (with various electron partners) also conforms to a double-displacement mechanism [75,76], although in this case the electronic route between the flavin

Fig 6 The forward and reverse reactions

catalysed by ferredoxin-NADP(H)reductase.

NADP + reduction (A) follows the

compul-sory ordered pathway of Fig 2, whereas two

alternative mechanisms are proposed for

electron transfer in the reverse direction:

ordered (B), or two-step transfer (C) E,

FNR ox ; F, FNR sq ; G, FNR red ; A, NADP +

P, NADPH; B, Fd ; Q, Fd

KdðNADPHÞKmðNADPHÞþ K0