Tài liệu Báo cáo Y học: Mutations in the docking site for cytochrome c on the Paracoccus heme aa3 oxidase Electron entry and kinetic phases of the reaction pptx

Mutations in the docking site for cytochrome c on the ParacoccusElectron entry and kinetic phases of the reaction Viktoria Drosou1, Francesco Malatesta2and Bernd Ludwig1 1 Molecular Gene

Mutations in the docking site for cytochrome c on the Paracoccus

Electron entry and kinetic phases of the reaction

Viktoria Drosou1, Francesco Malatesta2and Bernd Ludwig1

1 Molecular Genetics, Institute of Biochemistry, Johann-Wolfgang-Goethe Universita¨t, Frankfurt, Germany;

2

Department of Basic and Applied Biology, University of L’Aquila, Italy

Introducing site-directed mutations in surface-exposed

residues of subunit II of the heme aa3cytochrome c oxidase

of Paracoccus denitrificans, we analyze the kinetic

para-meters of electron transfer from reduced horse heart

cyto-chrome c Specifically we address the following issues: (a)

which residues on oxidase contribute to the docking site for

cytochrome c, (b) is an aromatic side chain required for

electron entry from cytochrome c, and (c) what is the

molecular basis for the previously observed biphasic reaction

kinetics From our data we conclude that tryptophan 121 on

subunit II is the sole entry point for electrons on their way to

the CuAcenter and that its precise spatial arrangement, but not its aromatic nature, is a prerequisite for efficient electron transfer With different reaction partners and experimental conditions, biphasicity can always be induced and is critically dependent on the ionic strength during the reaction For an alternative explanation to account for this phenomenon, we find no evidence for a second cytochrome c binding site on oxidase

Keywords: Paracoccus denitrificans; cytochrome c oxidase; docking site; electron transfer; biphasic kinetics

Cytochrome c oxidase is the terminal complex of the

respiratory chains of mitochondria and many bacteria

[1–4] It catalyzes the reduction of oxygen to water, coupling

the free energy of this reaction to the generation of a proton

gradient across the membrane

During the redox reaction, an electron delivered from

cytochrome c is first transferred to CuA, a binuclear copper

center located close to the surface of the large hydrophilic

domain of subunit II It is then donated to heme a

embedded in subunit I, and subsequently to the heme a3Æ

CuB center where oxygen reduction, and most likely the

redox coupling to proton pumping, take place

While the mitochondrial enzyme comprises up to 13

different subunits in a dimeric complex, the oxidase of the

bacterium Paracoccus denitrificans consists of only four

subunits, with the three largest ones homologous to the

corresponding mitochondrial subunits

Typically, many isolated oxidases are somewhat

promis-cuous towards their substrate molecules Early studies

analyzing the surface properties of cytochromes c of

different origin revealed a basic cluster of mostly lysine

residues located around the heme crevice Being responsible

for docking to their redox partners, an interaction between

cytochrome c and oxidase based on electrostatic forces was described [5–7] Experiments with monoclonal antibodies directed against subunit II of cytochrome c oxidase lead to a loss of activity [8] and supported the notion that the catalytic binding site is located predominantly on subunit II This result was confirmed by chemical modifications and early site-directed mutagenesis experiments [9,10], and is consis-tent with the crystal structures of the eukaryotic and the Paracoccus denitrificansoxidase showing clusters of negat-ively charged residues on the surface of subunit II [11,12] Previous studies on the binding of cytochrome c to the Paracoccusoxidase were interpreted by a two-step model in which electrostatic forces are responsible for an efficient long-range docking, followed by the reorientation of the redox partner driven by hydrophobic interactions [13] Specifically, a set of four acidic residues exposed on subunit

II (D135, D178, and to a lesser extent, E126, D159; see Fig 1 and [13]) had been assumed to interact electrostat-ically with the horse heart cytochrome c While a pivotal role in electron transfer from cytochrome c was assigned to residue W121 on subunit II [14], this early study did not address the question whether any other (aromatic) side chains in this or the neighbouring position might be able to support electron transfer to the CuAsite, thereby function-ally replacing tryptophan 121

As already observed previously, oxidase kinetics may yield nonlinear Eadie–Hofstee plots (e.g [15]) Two different phases, denoted high and low affinity, are clearly discern-ible, each being characterized by a set of individual kinetic parameters These biphasic steady-state kinetics become monophasic at higher ionic strength, a phenomenon discussed in terms of different binding sites (e.g [15,16])

or of conformational changes within the enzyme-substrate complex related to the coupling of electron transfer with proton pumping [17]

Correspondence to B Ludwig, Molecular Genetics, Institute of

Biochemistry, Biozentrum, Marie-Curie-Strasse 9,

D-60439 Frankfurt, Germany.

Fax: + 49 69 798 29244, Tel.: + 49 69 798 29237,

E-mail: ludwig@em.uni-frankfurt.de

Abbreviations: I, ionic strength; c 552 -f: soluble fragment of the

Paracoccus denitrificans cytochrome c 552 , expressed and purified from

Escherichia coli.

(Received 4 February 2002, revised 12 April 2002,

accepted 2 May 2002)

Here we give a comprehensive description of the

inter-action domain for cytochrome c on subunit II of the

P denitrificans oxidase, specifically addressing (a) the

electron entry point to oxidase and its possible alternatives,

(b) the size and extent of the acidic patch on subunit II, and

(c) ways of experimentally influencing the kinetic phases

during enzyme turnover

M A T E R I A L S A N D M E T H O D S

Mutagenesis and enzyme preparation

Site-directed mutagenesis in the ctaC gene was

per-formed according to the altered site mutagenesis protocol

(Promega, Heidelberg) Complementation of the

oxidase-deficient deletion mutant ST4 was performed as described

previously [18] Mutant strains were grown aerobically in

succinate medium [19] including streptomycin sulfate

(25 lgÆmL)1), membranes isolated according to [20] and

solubilized using n-dodecyl b-D-maltoside

The four-subunit cytochrome c oxidase was purified by

conventional chromatographic protocol as described in [21];

the oxidase complexed with an antibody fragment (Fv) was

isolated in a single chromatographic step as described

previously [22,23], and excess Fvremoved by gel filtration

The two-subunit oxidase complex was prepared by

combining the standard purification in dodecyl maltoside

[21], however, running the gel filtration step in the presence

of Triton X-100 to dissociate subunits III and IV [24]

Briefly, the following steps were performed: The

superna-tant after ultracentrifugation was loaded on the first

column, a DEAE-Sepharose CL-6B (Pharmacia Biotech)

equilibrated with 20 mM potassium phosphate pH 8.0,

1 mMEDTA and 0.5 gÆL)1dodecyl maltoside For elution,

a linear gradient ranging from 100 to 600 mM NaCl was used with this buffer Fractions of highest heme/protein ratio were pooled and concentrated in an Amicon cell (cut off 30 kDa) Triton X-100 was added to a final concentra-tion of 10% (w/v) The soluconcentra-tion was stirred for 1 h at 4C, applied to an Ultrogel AcA34 (IBF Biotechniques) gel filtration column equilibrated and processed with the above buffer with 2 gÆL)1 Triton X-100 replacing the dodecyl maltoside To reintroduce this latter detergent for subse-quent steps (and to avoid the detrimental effects of Triton X-100 on activity [25]), pooled fractions were rechromato-graphed on the first column equilibrated in 0.2 gÆL)1 dodecyl maltoside, and eluted with a linear gradient from

100 to 400 mMNaCl The oxidase fractions were analyzed

by SDS/PAGE to verify their subunit composition, con-centrated and stored at)80 C

Steady-state kinetics and determination of the ionic strength dependence

Cytochrome oxidase activity was measured with a Kontron Uvikon 941 spectrophotometer at 25C in 20 mM Tris/ HCl, pH 7.5, 1 mMEDTA, 0.2 gÆL)1n-dodecyl b-D -malto-side The different ionic strength conditions were adjusted

by adding KCl Ferrocytochrome c (horse heart, Sigma) was prepared by reduction with dithionite and excess reductant removed by Sephadex G25 chromatography Concentrations were varied between 0.5 and 40 lM, and oxidation followed at 550 nm after adding the purified enzyme (40 p up to 400 p ) Determination the

ionic-Fig 1 The presumed docking site for

cyto-chrome c on subunit II of the P denitrificans

oxidase The periplasmically oriented

hydro-philic domain housing the homodimeric Cu A

site (blue spheres) is depicted, omitting most of

the two transmembrane helices (bottom).

Selected side chains shown in detail were

mutated; the residue crucial for electron entry,

W121, is highlighted in yellow, along with

other residues important for docking The

figure was prepared on the basis of the

pub-lished coordinates (pdb1ar1), using the SWISS

PDB VIEWER/POV RAY program [31].

strength (I) dependence was performed in the same buffer at

20 lM cytochrome c, with the ionic strength adjusted to

values between 1.8 mMand 296 mMby the addition of KCl

The buffer for I¼ 1.8 mMwas 2.5 mMTris/HCl, pH 7.5,

0.2 gÆL)1dodecyl maltoside

Stopped-flow kinetics

The presteady-state kinetics were followed on a

ther-mostated Applied Photophysics DX.117 MV

stopped-flow apparatus at 20C in 20 mM potassium phosphate

pH 7.6, 1 mM EDTA, 0.2 g L)1 dodecyl maltoside The

ionic strength was 140 mM, adjusted with KCl Cytochrome

oxidase (4–6 lM) was incubated with 5 mMKCN at 4C

for at least 6 h Cytochrome c concentrations were varied in

the range of 2–32 lM; after mixing, the reaction was

followed at 550 nm (oxidation of cytochrome c) and/or

605 nm (reduction of heme a), and biphasic time courses

were obtained Three independent measurements were done

for each donor concentration, and the average was fitted to

the sum of two exponentials The observed rate constant

from the fast phase of this double-exponential decay was

plotted against the cytochrome c concentration, and the

apparent bimolecular rate constant koncalculated from the

slope

R E S U L T S

The large, periplasmically oriented hydrophilic domain of

subunit II of the heme aa3 oxidase represents the major

interaction site for cytochrome c We constructed a set of

mutants in surface-exposed residues to further identify

amino acid side chains involved in the docking of

cytochrome c, or in the presumed electron entry from

cytochrome c to the CuA center Positions subjected to

mutagenesis in this and two previous studies [13,14] are

summarized in Fig 1 All mutant enzymes, and several

preparations of oxidase differing in polypeptide

composi-tion, were assayed for their kinetic parameters under

different ionic strength conditions

Ionic strength dependence of the turnover number

The reaction of cytochrome c oxidase shows a strict

dependence of the turnover number on ionic strength We

measured the oxidation of 20 lMhorse heart cytochrome c

by the various oxidase preparations under steady-state

conditions, all yielding bell-shaped curves While the

optimum ionic strength for the wild-type enzyme was found

to be 56 mM(Fig 2; see also [13]), those for the two-subunit

enzyme (Fig 2) and all mutants in positions 121 and 122

(W121F, W121Y, W121Q/Y122Q, W121F/Y122F, W121G

and W121Y/Y122W) were decreased to 36 mM Of the

remaining mutants, two showed wild-type behaviour

(H119N, N160D), whereas all others were shifted to

46 mM(data not shown)

Turnover and presteady-state kinetics

We measured steady-state kinetics for all the subunit II

mutants at their optimum ionic strength as determined

above, using the reduced horse heart cytochrome c The

46 m ionic strength group was assayed both at 36 and at

56 mM, to allow for an unequivocal assignment to either a hyperbolic or nonhyperbolic Michaelis–Menten kinetic regime Tables 1 and 2 list the relevant parameters, Km and kcat, for the different ionic strength conditions: the Km value is taken from the so-called high-affinity phase, and the

kcatvalue is derived from the low-affinity phase

Positions W121 and Y122 A comprehensive set of single mutants in each of the two positions, or of double mutants, was generated (Table 1) While Kmvalues for all complexes

do not deviate from that of wild-type by more than a factor

of 1.6, the catalytic activity of any mutant in the W121 position is drastically diminished Residual activities for the two nonaromatic replacements (Q, G) are between 1 and 2% Additional single mutations in the 121 position which exchange the tryptophan for two other aromatic residues (F, Y) show almost the same distinct loss of electron transfer activity, with residual kcatvalues around 3–5% compared to wild-type When the neighbouring Y122 residue is changed

to a phenylalanine, the kinetic behaviour is that of wild-type, and a glutamine in this position only reduces activity

to 50% We conclude that Y122 is not involved in the

Fig 2 Ionic strength dependence of the turnover number for the isolated four- and two-subunit Paracoccus heme aa 3 oxidase complex The spectrophotometric assay was performed under steady-state condi-tions with 20 l M horse heart cytochrome c; for further details, see Materials and methods 4 su, four-subunit; 2 su, two-subunit complex.

Table 1 Steady-state and stopped-flowparameters of horse heart cytochrome c oxidation by Paracoccus oxidase mutated in selected exposed aromatic residues of subunit II The K m value was taken from the high-affinity phase at I ¼ 36 m M The k cat value was taken from the low-affinity phase at I ¼ 36 m M NR, no rate measurable.

Mutant position K m (l M ) k cat (s)1) k on · 10 6

( M )1 Æs)1)

Wild-type oxidase 1.4 669 3.7

W121Y/Y122W 1.2 7 0.13

electron transfer from cytochrome c to any large extent, nor

is this position involved in maintaining the low residual

activity when the W121 residue is mutated Double mutants

in both positions are not further diminished in activity

compared to W121 single mutations (see Table 1) Further

mutations (Y226F, H119N) in residues previously

consid-ered as potential alternative entry points for electrons from

cytochrome c (see Discussion) showed no deviations from

wild-type in their kinetic properties

To exclude the possibility that diminished electron

transfer activities in turnover experiments might be due to

changes in redox properties of the first acceptor in oxidase,

CuA, we measured relevant redox steps in the W121F

mutant, confirming that the redox potential for CuAis in the

wild-type range (P Hellwig, Institut fu¨r Biophysik,

Johann-Wolfgang, Goethe Universita¨t, Frankfurt, Germany,

personal communication)

Focussing on the parameters in Table 2 we found an

increase of Kmfor mutants H119I/Q120I, D146N, E140Q

and P196G measured at 56 mM Comparing these values

with those measured at 36 mM, again we found increased

Kmvalues for these mutants and also for E142Q kcatas the

parameter for maximum turnover is decreased Mutants

H119N and N160D reveal wild-type values; these positions

do not seem to be involved in cytochrome c binding

We also assayed the oxidase mutants under

presteady-state conditions, to ensure that the observed effects indeed

relate to the early phases of electron entry Using the

cyanide-inhibited enzyme, the reaction sequence is limited to

the transfer of the first two electrons reaching the CuA/heme

a redox couple To shift cytochrome c oxidation kinetics

into the time resolution of a stopped-flow apparatus, the

reaction was followed at 140 mM ionic strength (see

Materials and methods) and recorded at 550 nm (oxidation

of cytochrome c) and at 605 nm (reduction of heme a) The

observed time course was described by a sum of two

exponentials The fitted pseudo-first order rate constant was

plotted against the cytochrome c concentration after

mix-ing From the slope of this linear plot the bimolecular rate

constants konwere calculated for the wild-type and mutant

enzymes (Tables 1 and 2) This analysis reflects and

con-firms the kcatvalues obtained from turnover experiments Some of the mutants showed extremely slow reduction behaviour, and bimolecular rates could not be determined (see Table 1) The kon values for the mutants Y122F, Y226F, N160D, H119N and H119I/Q120I are in the same range as the wild-type oxidase while the other mutants show

a significantly decreased konvalue (see Tables 1 and 2)

Kinetic differences between the two-subunit and the four-subunit wild-type and mutant complexes

To assess kinetic properties of both forms under identical detergent conditions, we prepared cytochrome c oxidase lacking both subunits III and IV by replacing the detergent

in one of the chromatographic steps of the standard purification procedure (see Materials and methods): prior

to gel filtration, the partially purified material was incubated with a large excess of Triton X-100, known to dissociate the oxidase and leave an enzymatically active two-subunit complex [24] After gel filtration in Triton, dodecyl malto-side was reintroduced in the final step of column purification

to exclude known detergent effects in the subsequent analysis [25]

Ionic strength dependency of the maximum turnover number was shifted from 56 mM for the four-subunit complex to 36 mM for the two-subunit preparation Figure 2 also demonstrates that both complexes display a basically similar line shape, and turnover numbers are in close agreement at 20 lMcytochrome c This behaviour is taken as a first evidence that the periplasmically oriented regions of one or both of the two ancillary subunits may contribute to some extent to the interaction domain for the substrate (see also Discussion)

Kinetic parameters for both complexes under several ionic strength conditions are listed in Table 3 Comparing

Kmand kcateach at optimum ionic strength for both forms,

it is evident that kcatis lower by a factor of three for the two-subunit enzyme, while its Kmis diminished twofold The overall specificity constant (kcat/Km) of this two-subunit complex therefore remains in the same range, explaining in part its comparable activity at a given substrate

concentra-Table 2 Oxidation of horse heart cytochrome c by wild-type oxidase and subunit II mutants under turnover and pre-steady state conditions at different ionic strengths TM1, triple mutant (E126Q, D135N, D178N) in subunit II ND, not determined.

Mutation K m (l M ) k cat (s)1) K m (l M ) k cat (s)1) k on · 10 6 ( M )1 Æs)1) b

a Mutants as published in [13] were re-analyzed side by side, and are presented for a complete overview b K m value taken from the high-affinity and k cat from the low-affinity phase c Pre-steady-state kinetics of cytochrome c oxidation measured by stopped-flow, see Materials and methods for details.

tion (Fig 2) Analyzing lower ionic strength datasets for

both preparations, the general trend persists that kcat is

below that of the four-subunit enzyme, while Km values

approach each other (see Table 3)

Shifts from mono- to biphasic behaviour are observed for

both the four-subunit and the two-subunit oxidase on going

from higher ionic strength to lower values Figure 3

exemplifies this transition to nonlinear kinetic behaviour

for the two-subunit complex when [I] is diminished in steps

from 56 to 15 mM Eadie–Hofstee plots yield clear breaks

for the two lower salt conditions (Fig 3B) These transition

points are listed in Table 3 (last column) for selected

preparations/mutants (see also below) Also this criterion

distinguishes the two-subunit variant from the four-subunit

complex, where the transition occurs already at 36 mM,

clearly indicating that biphasic kinetics are not due to the

presence of subunits III and IV

Biphasic behaviour of the triple mutant TM1 (subunit II:

E126Q, D135N, D178N) is evident when the four-subunit

complex is assayed: while other mutants containing single

acidic residue replacements followed biphasic kinetics at

I¼ 36 mM (not detailed), TM1 was monophasic at

I¼ 36 mM Nevertheless, on further decreasing ionic

strength, biphasic kinetics were again observed with a

transition point at around 15 mM(see Table 3) The same

holds true when the TM1 preparation was stripped of its

subunits III and IV: the resulting two-subunit mutant

complex displayed biphasic kinetics at 15 mM ionic

strength

Further criteria for manipulating the kinetic phases

of reaction

Transitions from mono- to biphasic reaction conditions,

depending on ionic strength variation, can be induced by

other means as well Specific F fragments, derived from

Table 3 Kinetic parameters and biphasic transitions under different ionic strength conditions for selected oxidase preparations ND, not determined.

I ¼ 7.4 m M I ¼ 14.8 m M I ¼ 26 m M I ¼ 36 m M I ¼ 56 m M

Biphasicity

Oxidase preparation

K m

(l M )

k cat

(s)1) a

K m

(l M )

k cat

(s)1)

K m

(l M )

k cat

(s)1)

K m

(l M )

k cat

(s)1)

K m

(l M )

k cat

(s)1)

transition at

I (m M ) b

Four-subunit oxidase ND ND 0.6 434 0.9 555 1.4 669 5.9 1031 36

Four-subunit oxidase ND ND 0.3 63 1.6 154 3.6 338 10.5 400 26

purified with F v

Four-subunit oxidase ND ND 0.5 88 1.1 270 4.1 384 ND ND 26

+ specific F v added

Four-subunit oxidase ND ND ND ND ND ND 1.6 555 ND ND 36

+ control F v added

Two-subunit oxidase ND ND 0.8 254 0.85 263 2.9 288 15.1 336 26

Two-subunit oxidase 1.6 220 3.8 243 ND ND ND ND ND ND 7.4 + specific F v added

Four-subunit TM1c ND ND 0.56 8 1.6 20 7.9 25 ND ND 14.8

Four-subunit oxidase ND ND 2.8 1000 28.5 474 52.5 100 ND ND 26

vs c 552 – fd

a The K m value is taken from the high-affinity phase and k cat from the low-affinity phase, whenever kinetics are biphasic b On lowering the ionic strength, transition from monophasic to biphasic kinetics is observed at specified ionic strength (I).cTriple mutant TM1 (E126Q, D135N, D178N) in subunit II.dData taken from V Drosou & B Ludwig, unpublished results.

Fig 3 Eadie–Hofstee plots (A and B) representing horse heart cyto-chrome c oxidation by the two-subunit oxidase at different ionic strength conditions Steady-state kinetics were determined spectrophotometri-cally at 25 C.

monoclonal IgG directed against a subunit II epitope [22],

may be added in a 3 : 1 molar excess to purified oxidase

both as a four- or a two-subunit complex Alternatively, Fv

may be used to affinity-purify the four-subunit oxidase from

solubilized membranes, yielding a stable 1 : 1 complex

which was instrumental in the structure determination of

the P denitrificans oxidase [11] Table 3 indicates that in all

cases the Fvfragment, present with or added to the enzyme,

induced a decrease in the transition point to biphasic

kinetics To some extent, individual effects appear to be

additive when following this shift from the four-subunit to

the two-subunit enzyme, and to the Fv-complexed oxidase

lacking the two smallest subunits

In a control reaction employing an unspecific Fvprotein

not recognizing any oxidase epitope [26], wild-type

beha-viour ensued It should also been noted that under true

biphasic conditions (26 mM), kinetic parameters for the

Fv-complexed oxidase point at a somewhat diminished

overall catalytic efficiency of this enzyme form (see Table 3),

although, with the exception of the Fv-complexed

two-subunit oxidase, the high affinity Kmvalues are comparable

While all the above mentioned experiments were

per-formed with the commercially available horse heart

cyto-chrome c, the heterologous expression of a soluble c-type

cytochrome fragment, c552-f, will allow to probe this

bacterial oxidase with its homologous electron donor

derived from P denitrificans [27–30] With regard to

reaction kinetics with the four-subunit oxidase complex,

this soluble bacterial cytochrome fragment is a competent

donor to oxidase (V Drosou & B Ludwig, unpublished

results), and more importantly it is characterized by biphasic

Eadie–Hofstee plots once the ionic strength drops to 26 mM

or below (see Table 3, last row)

D I S C U S S I O N

Extent of the acidic patch on subunit II involved

in the cytochromec docking reaction

A two-step model has been proposed to describe the docking

of the membrane-embedded oxidase with its soluble

sub-strate cytochrome c In a first step governed by long–range

electrostatic interaction mediated by oppositely charged

surfaces on either protein, a preorientation of both redox

partners is obtained, which is followed by a fine-tuning

mediated by hydrophobic surfaces to aquire a docking

conformation for optimal electron transfer [14,32] A strong,

positive surface potential for the mitochondrial electron

donor, cytochrome c, is evident, while several acidic residues

have been suggested to participate in docking on a negatively

charged patch located mostly on subunit II above the first

electron acceptor in oxidase, the CuAcenter (see

introduc-tion) A bell-shaped dependency of the turnover number on

ionic strength of the assay medium (see also Fig 2) has been

taken as initial experimental evidence that protein surface

charges get progressively shielded by increasing the ionic

strength of the medium Under turnover conditions, an

optimal salt concentration results from a compromise of the

association and the dissociation rates for cytochrome c

From a previous mutagenesis study [13], a partial

contribution of a few acidic residues on subunits I and III

to the acidic docking site on the periplasmic face of the

P denitrificansoxidase appeared likely Making use of the

fact that this bacterial enzyme can be isolated both as a four- and a two-subunit complex without major kinetic defects (see Fig 2, and below), a distinct decrease (by

20 mM) in the ionic strength maximum for the two-subunit wild-type complex confirms the contribution of additional charge(s) located on the two further subunits of the native oxidase

In focussing on the main interaction domain on subunit

II, we introduced additional mutations in exposed residues

in the relevant area above the CuA site (see Fig 1 and Table 2), to estimate the extent of the acidic region responsible for cytochrome c docking While no direct structural information is at hand for the docked complex, the interaction domain for cytochrome c on the cyto-chrome bc1complex of yeast turned out to be confined to a few residues only [33]

Both the mutants H119N and N160D (Table 2) show wild-type characteristics, along with an ionic strength optimum at 56 mM For H119N this is not surprising since

no charge change results In position N160 an additional negative charge was introduced, but available kinetic parameters suggest that this mutant, despite its higher negative surface potential, does not provide a more potent docking site for its substrate This observation may be explained by the fact that this residue is located too far out from the actual electron entry site W121 (see below) Mutants E140Q, E142Q, D146N, and P196G all show a shift in the ionic strength optimum to 46 mM, providing first evidence that these residues are involved in substrate binding They were characterized at 56 mMand at 36 mM;

in the latter condition, clear biphasic kinetics were recorded (see below) Mutants D135N and D178N and the triple mutant TM1 have already been characterized as bona fide docking mutants in the past [13] but were re-analyzed side by side with the other mutants generated here Three of these positions were changed from an acidic side chain into the corresponding amide derivative, yielding unequivocal evi-dence for their contribution in the cytochrome c oxidation reaction Compared to wild-type, they show some changes

in Km, but at the same time also in kcat When biphasic reactions are obtained at 36 mMionic strength, the tendency increases for a more pronounced rise in the Kmvalue, but a concomitant loss in kcatcannot be overlooked under this condition either This decrease of turnover numbers may partly be explained by the fact that for the purpose of a uniform comparison, these mutants were measured at

36 mM and 56 mM whereas the individual optimal ionic strength was found to be at 46 mMin some cases

When presteady-state kinetics are measured at 140 mM

ionic strength for this set of mutants, it is evident that a parallel trend, even though not always in a quantitative manner, is seen for E142, D146N, and E140Q (Table 2) The double mutant H119I/Q120I should lead to an increase of the hydrophobic free energy, and its Kmvalue is increased (along with a decrease of kcat), which means that substrate binding is influenced Since the single mutant H119N showed wild-type behaviour, the position Q120 is most likely responsible for the observed effects

The interpretation of the low-affinity Km values (not given) is not straightforward since the explanation for this phase is still hypothetical (see below) However, the same general trend for both the high-affinity and the low-affinity

K values is observed

Taken together with our earlier data [13], this study now

defines an extensive area of exposed acidic residues on

subunit II which are involved in the initial docking (see

above) of the horse heart cytochrome c In viewing down

the axis from W121 to the CuAcenter as in Fig 1, a lobe of

three carboxylate side groups (D146, E140, D159), with a

minor contribution from E142, extends to the edge of the

presumed interaction site A more central region, closer to

W121, is made up of residues D135, E126, and D178 (as

modified together in the triple mutant TM1) Further

residues important for interaction in this latter lobe include

Q120, and possibly P196 This docking site model includes

the four homologous positions of acidic residues considered

most effective also in the Rhodobacter spheroides heme aa3

oxidase [34]

Experiments replacing the mitochondrial cytochrome c

with a fragment of the homologous bacterial electron

donor, cytochrome c552of P denitrificans [27,28] confirm

that all of the above mentioned residues on oxidase are also

involved in this docking reaction, while some additional

ones appear specific for the bacterial donor protein (for

details, see V Drosou & B Ludwig, unpublished results)

From this we conclude that the surface area on oxidase,

covered by the bacterial cytochrome c, is at least as large as

that for the mitochondrial protein

Specificity of the electron entry site into oxidase

Previous mutagenesis data on the P denitrificans [14] and

on the Rh spheroides [34] oxidase clearly indicated that the

tryptophan at position 121 is of crucial importance for

electron transfer from cytochrome c to the CuAcenter in

oxidase Being located approx 5 A˚ above the metal

center, it is followed in sequence by another aromatic side

chain, Y122 Table 1 summarizes the kinetic effects of

single mutations in either residue, and of several double

mutants, indicating that in no case any major changes on

Km, resp., on affinity towards the substrate, occur

However, whenever a W121 mutation is introduced, kcat

is drastically diminished to a few percent residual activity

for aromatic side chain replacements, and even lower for

aliphatic ones On the contrary, exchanges in the

neigh-bouring aromatic residue, Y122, only lead to moderate or

no activity changes at all Double mutants like the

W121Q/Y122Q do not fall below the single W121Q

activity, i.e its (already low) residual electron transfer

activity is not maintained by the neighbouring tyrosine,

while the W121F/Y122F mutant activity may be viewed as

a commitment of the tyrosine residue to support the

(somewhat higher) residual activity of the W121F single

mutant Pre-steady-state kinetics again fully support the

turnover data, showing that for some cases a bimolecular

rate in the electron transfer reaction is no longer

meas-urable (see Table 1)

We conclude that a tryptophan is strictly required in this

position to accept electrons from cytochrome c, most likely

for steric reasons, since virtually no other residue, not even

another aromate, neither in this position nor an adjacent

position, is apt for maintaining this role This statement

seems to hold true for further alternative positions suggested

from computational docking studies (L Dutton, Johnson

Foundation, Philadelphia, PA, USA, personal

communi-cation) as potential entry site: mutations in an exposed

tyrosine (Y226F; see Table 1) and in a histidine (H119N; Table 2) show wild-type kinetics

Biphasic steady-state kinetics Non-linear kinetics have been observed for cytochrome c oxidation (see introduction) in many experimental systems Generally speaking, higher ionic strength conditions result

in monophasic plots in a typical Eadie–Hofstee presenta-tion, whereas experiments at lower ionic strength may lead

to biphasic kinetics This effect is exemplified in Fig 3 for the isolated two-subunit oxidase complex in going from

I¼ 56 to I ¼ 15 mMionic strength assay conditions, where the transition to biphasicity occurs at 26 mM Based on this observation, we further examine the bacterial oxidase and specify a number of widely differing conditions (see Table 3)

to manipulate this transition point from mono- to biphasic behaviour

Subunit composition of the oxidase complex, as already discussed above in terms of ionic strength optimum of cytochrome c oxidation, is an experimental criterion for differentiation: the two-subunit complex reaction becomes biphasic at a lower salt concentrations when compared to the four-subunit enzyme (see Table 3)

Loss of charged (acidic) side chains, either in many single mutations or in the triple mutant TM1 (Table 3), down-shifts the transition considerably, also in the context of the above subunit criterion

Binding of Fvto the subunit II epitope has a profound effect on the transition As outlined in Table 3, this cannot

be explained by the purification method since this effect occurs both for a Fv (affinity chromatography protocol) preparation as well as for a conventionally isolated enzyme incubated with a threefold molar excess of Fvprior to the kinetic measurement Moreover, the effect is specific for the particular epitope/antibody, and cannot be mimicked by addition of a Fvantibody preparation lacking any oxidase affinity This kinetic phenomenon is difficult to rationalize since the epitope is located on a site of subunit II, opposite

of the presumed docking area for cytochrome c [11], and a direct competition with substrate therefore appears unlikely

We also note that both the Kmand the kcatof oxidase are appreciably perturbed under most conditions when the specific Fvis present At least two possible explanations may

be given at this point, either a slight conformational

freezing effect due to the tight Fv binding, or a general disturbance of the surface potential of the hydrophilic region of this subunit

Different donor molecules do cause such shifts as well Comparing the standard horse heart cytochrome c with the homologous bacterial donor, c552 (employed as a soluble fragment; Table 3, last line), the transition point is lowered for the four-subunit complex reacting with the Paracoccus donor

From the above collection of examples (which are largely descriptive in nature), it is evident that so far we cannot find any in vitro conditions under which cytochrome c oxidation proceeds in a strictly monophasic manner, apart from increasing ionic strength Whenever the ionic strength in the assay medium is adequately reduced, nonhyperbolic Michaelis–Menten kinetics can be obtained However, in this investigation we have been able to exclude that this general feature depends on (a) the presence of subunits III

and IV in the Paracoccus enzyme, and by inference on the

presence of cytoplasmically coded subunits of the

mito-chondrial enzyme as well, and (b) on differences in

purification strategies We also have no evidence that

biphasicity is a consequence of a potential second binding

site for cytochrome c, as recently again suggested for the

mitochondrial enzyme on the basis of crosslinking

experi-ments [35] and theoretical considerations [36], which,

however, are in contradiction to early evidence, obtained

on spectroscopic grounds [37], favouring a single functional

binding site: Several attempts to eliminate a hypothetical

second site have been made here for the bacterial enzyme by

stripping subunits III and IV off the native complex, and by

further destroying a large part of the acidic lobe(s) of the

docking site of subunit II in the TM1 mutant Nevertheless,

even the latter construct, as a severely crippled two-subunit

complex, displays biphasic kinetics

Inspecting all the above data, it appears that the

transition point (to biphasic behaviour), as a general trend,

lies below the ionic strength value for the turnover

maximum We may speculate that the kinetic phenomenon

of biphasicity is simply caused, in mechanistic terms, by

steric interference between oxidized cytochrome c (with a

sluggish off-rate to dissociate from the enzyme), and the

next incoming reduced cytochrome c molecule, both

com-peting for the docking site under turnover conditions [15] In

this context it is interesting to note that even for a covalently

linked cytochrome c domain, as present, e.g in the caa3

oxidase of B subtilis, biphasic reaction kinetics have been

reported in the ascorbate/tetramethyl-p-phenylenediamine

assay [38] Thus, the observed low ionic strength

nonhy-perbolic Michaelis–Menten kinetics may not be solely due

to changes in the initial ferrocytochrome c concentration,

and rather are an intrinsic enzymic property ensuing from

the mechanistic details of the cytochrome oxidase reaction

A C K N O W L E D G E M E N T S

We are grateful to Maurizio Brunori and Oliver Richter for helpful

criticism, to Andrea Hermann and Hans-Werner Mu¨ller for excellent

technical assistance, and thank Petra Hellwig for help with the Cu A

redox potential determination This work was supported by Deutsche

Forschungsgemeinschaft (SFB 472) and Fonds der Chemischen

Industrie, by Conferenza dei Rettori delle Universita` Italiane, and

Deutscher Akademischer Austauschdienst (DAAD Vigoni Program).

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