Báo cáo khoa học: Evidence for two different electron transfer pathways in the same enzyme, nitrate reductase A from Escherichia coli potx

Evidence for two different electron transfer pathways in the sameRoger Giordani* and Jean Buc Laboratoire de Chimie Bacte´rienne, Institut Fe´de´ratif ‘Biologie Structurale et Microbiolo

Evidence for two different electron transfer pathways in the same

Roger Giordani* and Jean Buc

Laboratoire de Chimie Bacte´rienne, Institut Fe´de´ratif ‘Biologie Structurale et Microbiologie’, Centre National de la Recherche Scientifique, Marseille, France

In order to clarify the role of cytochrome in nitrate reductase

we have performed spectrophotometric and stopped-flow

kinetic studies of reduction and oxidation of the cytochrome

hemes with analogues of physiological quinones, using

menadione as an analogue of menaquinone and

duro-quinone as an analogue of ubiduro-quinone, and comparing the

results with those obtained with dithionite The

spectropho-tometric studies indicate that reduction of the cytochrome

hemes varies according to the analogue of quinone used, and

in no cases is it complete Stopped-flow kinetics of heme

oxidation by potassium nitrate indicates that there are two distinct reactions, depending on whether the hemes were previously reduced by menadiol or by duroquinol These re-sults, and those of spectrophotometric studies of a mutant lacking the highest-potential [Fe-S] cluster, allow us to pro-pose a two-pathway electron transfer model for nitrate reductase A from Escherichia coli

Keywords: cytochrome b; electron transfer; Escherichia coli; nitrate reductase A; quinone

Nitrate can be used as an electron acceptor for anaerobic

growth of Escherichia coli [1,2] This oxidoreduction is

catalysed by nitrate reductase A (EC 1.7.99.4) This enzyme

is a membrane-bound complex of three subunits, designated

a, b and c, coded by three genes, narG, narH and narJ,

which form a single operon [3–5] The a and b subunits are

located on the cytoplasmic side of the membrane and form

an ab complex that binds to the membrane by interacting

with the c subunit, a very hydrophobic protein embedded

in the membrane [ 4]

Each subunit carries a different set of redox centers [5,6]

The 139 kDa a subunit contains the active site for the

reduction of nitrate to nitrite and a molybdenum cofactor

(molybdopterin guanine dinucleotide); a recent

crystallo-graphic study has revealed the presence of a new [4Fe-4S]

cluster [7] in the a subunit The 58 kDa b subunit contains

four [Fe-S] clusters which belong to two classes: a

high-potential and a low-high-potential class The high-high-potential class

contains [4Fe-4S] and [3Fe-4S] clusters with redox potential

of +180 mV and +130 mV, respectively Redox potentials

of)55 and )420 mV correspond to two [4Fe-4S] clusters in

the low-potential class The 20 kDa c subunit is a

hydro-phobic cytochrome of type b carrying two hemes [8–10]

The electron transfer in membranous enzyme requires

a quinone as an electron donor, and either ubiquinone

(a benzoquinone) or menaquinone (a naphthoquinone) can

fulfil the role [11] A steady-state kinetic study of nitrate reductase was carried out by Morpeth et al [ 12] Unfortu-nately, however, this study did not take account of the stoichiometry of the reaction and in consequence gave incorrect rate equations and led to hazardous conclusions

A previous kinetic study [13] suggested that duroquinol (a ubiquinone analogue) and menadiol (a menaquinone analogue) deliver their electrons at two different sites on the nitrate reductase The loss of the highest-potential [4Fe-4S] cluster in a mutant form of nitrate reductase results in an enzyme devoid of menadione activity, but still retaining duroquinone activity The existence of a specific site of reaction for each quinol, together with the differences in the effects on the two quinols produced by loss of an [Fe-S] cluster, suggested the possibility of two separate pathways for transfer of electrons from duroquinol and menadiol in nitrate reductase A [13]

EPR [9] and potentiometric [14] studies show the existence of two b type hemes in the c subunit, cyto-chrome b, of nitrate reductase A The data from these studies support the assignment of the axial ligands to the low-potential heme (bL) (Em,7¼ 20 mV) and to the high-potential (Em,7¼ 120 mV) heme (bH), respectively, located near the periplasmic and the cytoplasmic side of the membrane Correct insertion of the two hemes into the

c subunit requires anchoring to the ab complex [9,10] Moreover, a kinetic study by stopped-flow of nitrate reductase with menadione only [15], exhibits four kinetic phases in the reduction of the hemes by menaquinol According to the quinone type used, the two hemes of the cytochrome are probably involved in the transfer of electrons from the two specific binding sites and the two specific pathways suggested [13] for transport of electrons from the two quinol types to [Fe-S] clusters, molybdenum cofactor and nitrate To clarify the role of the hemes we have made spectral and kinetic studies of the reduction

of these hemes with various analogues of physiological

Correspondence to J Buc, Laboratoire de Chimie Bacte´rienne, 31

chemin Joseph-Aiguier, B.P 71, 13042 Marseille Cedex 20, France.

Fax: + 33 491 7189 14, E-mail: buc@ibsm.cnrs-mrs.fr

Abbreviations: HOQNO, 2-n-heptyl-4-hydroxyquinoline-N-oxide.

Enzyme: nitrate reductase A (EC 1.7.99.4).

*Present address: Faculte´ de Pharmacie, Universite´ de la Me´diterrane´e,

13385 Marseille Cedex 05, France.

(Received 17 March 2004, revised 8 April 2004,

accepted 14 April 2004)

quinones and compared the results with those obtained with

dithionite Oxidation of reduced hemes was followed after

addition of potassium nitrate In view of the rapidity of the

reduction and oxidation reactions, the kinetic studies needed

to be made with a stopped-flow apparatus

Experimental procedures

Reagents and chemicals

Menadione (2-methyl-1,4-naphthoquinone), and

duroqui-none (tetramethyl-p-benzoquiduroqui-none) were purchased from

Aldrich Juglone (5-hydroxy-1,4-naphthoquinone),

plumb-agin (5-hydroxy 2-methyl-1,4-naphthoquinone),

coen-zyme Q0 (2,3-dimethyl 5-methyl-p-benzoquinone) and

benzyl viologen were from Sigma All other chemicals were

of the highest grade of purity commercially available, and

were supplied either by Prolabo or Merck

Bacterial strains and plasmids

The strains used in this study were LCB2048

(DNRA,DNRZ) as strain devoid of nitrate reductase [16],

pVA700, pJF119EH(narGHIJ)Apr, as overexpressed

wild-type plasmid [17] and pVA700-C16,

pJF119EH(nar-GH[C16A]IJ)Apr, as plasmid lacking the high-potential

[4Fe-4S] cluster [17]

Growth conditions

Growth conditions were those described previously [17] All

strains were grown anaerobically at 37°C on TY medium

supplemented with glucose (2 gÆL)1) Expression of nitrate

reductase was induced by isopropyl thio-b-D-galactoside

(0.2 lM) (pVA700) The antibiotics ampicillin (50 mgÆL)1)

and chloramphenicol (10 mgÆL)1) were used

Preparation of subcellular fractions

Cells were harvested during the exponential phase of growth

and suspended in 100 mM potassium phosphate buffer

pH 6.6 and disrupted at 69 MPa by passage through a

French press The resulting suspension was centrifuged

(for 15 min at 18 000 g) to sediment unbroken cells The

supernatant was further centrifuged for 90 min at a

maxi-mum of 120 000 g, and the new supernatant was discarded

while the pellet was retained All of these procedures were

performed at 4°C The pellets, containing nitrate reductase

as a complete membrane-bound complex, were resuspended

in a small volume of buffer and stored at)80 °C until use

Quantification of nitrate reductase

The concentration of nitrate reductase was estimated by

reference to the percentage of enzyme in the total protein

This percentage was determined from the percentage of

nitrate reductase antigen present in nitrate reductase

membranous preparations measured by rocket

immuno-electrophoresis [18] as described previously [19] Proteins

were estimated by the technique of Lowry et al [20] using

bovine serum albumin as standard The amount of

over-expressed enzymes was about 10-times that in the parent

strain MC4100 With the plasmids used here, which express the genes stoicheiometrically, about 90% of the enzyme is membrane-bound (further details in [17])

Enzyme assays Nitrate reductase activity with benzyl viologen as substrate was measured spectrophotometrically [21] by nitrate-dependent oxidation of reduced benzyl viologen, with the precautions described previously [22]

Nitrate reductase activities with quinols as substrates were measured by a spectrophotometric method as des-cribed previously [22]

Spectra These were obtained with a Hitachi U-2000 spectro-photometer, thermostated at 37°C and connected to a personal computer All experiments were performed in

50 mM potassium phosphate buffer pH 6.6 A 1.6 mL quartz cuvette closed by a Teflon cap with a central hole was used to allow the different reagents to be added with

a microsyringe Thorough mixing in the cuvette was achieved by displacement of glass beads As the mem-branes were from strains with nitrate reductase over-expressed, amounts of membrane low enough to avoid turbidity could be used

Reduction of quinone analogues Quinone analogue solution (1.7 mL of 20 mM, in ethanol) were added to 70 mg of zinc powder and 60 lL of 5MHCl, after mixing and sedimentation of the zinc the reduced analogue solution could used for two or three hours This technique, used in organic chemistry [23], is very efficient and is easier to use than that with KBH4, which was previously employed in nitrate reductase assays [22] Stopped-flow kinetics

Stopped-flow kinetic measurements were made with a Hi-Tech Scientific FF61 apparatus (Salisbury, UK) con-nected to a personal computer The stopped-flow appar-atus was equipped with a 1 cm light-path quartz cuvette

To minimize oxygen leaks, the drive system was sub-merged in a thermostatically controlled circulating water bath at 37°C The stopped-flow system was thoroughly flushed with anaerobic buffer (50 mM potassium phos-phate, pH 6.6) immediately before the experiments were started Solutions were equilibrated to assay temperature before experiments To measure the absorbance at

560 nm, measurement of baselines were established at each wavelength with anaerobic buffer, and the values obtained were typically within 5% of those obtained from the absorbance spectra of the same solutions recorded on the Hitachi U-2000 spectrophotometer

Analysis of data

A personal computer was used to fit experimental data to appropriate equations by nonlinear least squares, using a Newton–Gauss algorithm [24]

Spectrophotometric studies with quinone analogues

Spectra were measured between 500 and 600 nm in order to

follow the redox state of the cytochrome by following the

characteristic peak at 560 nm

If one takes as reference the reduction by dithionite, a

nonphysiological reducing agent that reduces the enzyme

completely and nonspecifically (heme, [Fe-S] clusters and

molybdedum cofactor) one can observe in the overexpressed

wild-type strain, a difference between reduced and oxidized

spectra, according to whether the subsequent reductant is

menadione or duroquinone (Fig 1) In both cases the

maximum amplitude at 560 nm is less than that obtained

with dithionite, which seems to indicate that the reduction

of the hemes of the cytochrome varies according to the

analogue of quinone used, and in neither case is it complete

Use of a strain devoid of nitrate reductase (LCB 2048)

makes very clear the lack of any signal at 560 nm (Fig 1,

trace 4)

Reduction by analogues is performed by addition of

small quantities of reduced analogue until there is no further

change in signal To reduce quinone analogues, the reducing

agent used was KBH4followed by zinc Zinc in the presence

of HCl has been preferred, as it gives a more reliable and

stable reduction in one step with the advantage of not

diluting the reducing solutions, thus allowing the addition of

smaller quantities for the same reducing effect In any event,

the results obtained are equivalent whatever the mode of reduction of analogues of quinone used The reduction

of membranes with dithionite is performed by addition of dithionite powder

These experiments have been carried out using a range of concentrations in membrane at the limit of turbidity of the solution (0.193–2.09 lM) In all cases one obtains similar results (Fig 1) that show the variation of the amplitude of the signal at 560 nm between reduced and oxidized forms

by the nitrate according to the concentration in nitrate reductase with the three electron donors used (dithionite, menadiol and duroquinol)

In the concentration range used, the amplitude of absorbance reduced minus oxidized is linearly correlated with the concentration of nitrate reductase (Fig 2) whatever the reducing agent used, the correlation coefficients of least-square regressions being 0.98, 0.98 and 0.96 for dithionite, menadiol and duroquinol, respectively

One can determine the slopes of the three straight lines obtained with the three electron donors These slopes correspond to the differences in molecular extinction coefficients of reduced and oxidized forms of the hemes, and therefore to apparent molecular extinction coefficients corresponding to maximal reduction of the various electron donors: 0.069 lM )1Æcm)1 for dithionite, 0.059 lM )1Æcm)1 for menadiol and 0.031 lM )1Æcm)1for duroquinol These values have induced us to verify with other analogues whether there are differences in amplitudes of the peak at 560 nm between reduced and oxidized forms

Fig 1 Difference spectra of membranous nitrate reductase

Mem-branous enzyme was reduced by (1) dithionite, (2) menadiol and (3)

duroquinol Reductions were obtained by addition of small amounts

of reduced analogues or dithionite until no further change in the

spectrum Oxidations were performed by addition of nitrate Trace (4)

was obtained with a strain lacking nitrate reductase (LCB 2048) and

dithionite as reductant The membranous nitrate reductase

concen-tration was 1.2 l in all cases.

Fig 2 Variation of absorbance difference between reduced and oxidized membranous nitrate reductase vs enzyme concentration Membranous nitrate reductase was reduced by (1) dithionite, (2) menadiol and (3) duroquinol Lines were obtained by least-squares fitting, the slopes of these lines being consistent with the apparent molecular extinction coefficients The absorbances were measuered at 560 nm.

We have used analogues of type ubiquinone (lapachol

and Coenzyme Q0 and decylubiquinone), and analogues

of type menaquinone (plumbagine and juglone) These

studies have been undertaken with various enzyme

concentrations to permit us to obtain the apparent

molecular extinction coefficients (Table 1) Except for

decylubiquinone, which induces flocculation in the cuvette,

we obtained a difference of amplitude of the peak at

560 nm between the form reduced by analogues and the

form oxidized by nitrate, and in all cases this difference

was less than that obtained with dithionite The differences

of molecular extinction coefficients for reduced and

oxidized forms can be grouped into two classes according

to their values, the analogues of ubiquinone and those of

menaquinone, these last having larger differences of

coefficients than the first This is not surprising, because

menaquinols are the preferred electron donors for nitrate

reductase in anaerobic conditions [25]

These analogues (lapachol, plumbagine, etc.), unlike

menadiol and duroquinol, give spectra with significant

baselines in the absence of membrane, necessitating

corrections to the spectra In addition, the specific

absorption of analogues at 560 nm complicates the kinetic

study of reduction of the cytochrome according to the

choice of menadiol and duroquinol for spectral and

kinetic studies

Influence of HOQNO on spectra

The presence of the menaquinone analogue

2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) in the medium

inhib-its reduction of the cytochrome by menadiol, but does not

inhibit reduction by duroquinol (Fig 3) It is probable that

the nucleus of the HOQNO molecule, similar to that of

duroquinone, i.e a nucleus of type benzoquinone,

specific-ally inhibits the site for menadiol, as predicted by previous

studies showing cross-inhibition of menadiol and

duro-quinol on the binding of these electrons donors to the

cytochrome b of nitrate reductase [13]

These results with HOQNO explain those of Rothery

et al [23], who found a site of interaction between the

cytochrome and the quinones by studying inhibition of the

nitrate reductase activity by HOQNO It has therefore

clarified the binding site of the menaquinols, but for technical

reasons it cannot reveal the site for the ubiquinols, because

HOQNO does not inhibit binding of the ubiquinols

Kinetic studies by stopped-flow

We have studied oxidation by nitrate of the cytochrome

of nitrate reductase, preliminarily reduced by an analogue

of quinone, menadiol or duroquinol We have made experiments at various concentrations of electron donors and nitrate, in each case for several ranges of time (0.5–5 s) The traces obtained are shown in Fig 4 They are monophasic and fit a decreasing exponential equation The amplitudes obtained agree with those obtained during spectrophotometric studies (Fig 1) The time constants corresponding to apparent rate constants are obtained by fitting Eqn (1):

where, DAbs is the variation of absorbance, a and b are amplitude parameters and c is the time constant

The time constants are different according to whether the enzyme has been reduced by menadiol or by duroquinol; the results are listed in Table 2

Whatever the experimental conditions, the apparent rate constant is 1 s)1 with menadiol, whereas it is 2 s)1with duroquinol This doubling of the apparent rate constants indicates that there are two distinct reactions, and so oxidation of the cytochrome by nitrate follows separate pathways according to the nature of the electron donor The kinetics of reduction of the cytochrome is very complex In time ranges from 0.5 to 10 s, one observes traces corresponding to the sum of three exponentials at least These traces are too complex to be analysed with precision The residual plots, difference between experimental data and calculated values obtained after fitting, shown in Fig 4,

Table 1 Apparent molecular extinction coefficients between reduced

and oxidized membranous nitrate reductase for different reductants.

e values were obtained by least-squares fitting, like those presented in

Fig 2.

Reductant E m (mV) e (l M )1 Æcm)1)

Artificial reductant Dithionite 0.069 ± 0.002

Menaquinone Menadione )1 0.059 ± 0.004

analogues Plumbagine )74 0.043 ± 0.001

Juglone +33 0.037 ± 0.005 Lapachol )179 0.046 ± 0.005 Ubiqinone Duroquinone +35 0.031 ± 0.04

analogues Coenzyme Q 0 +10 0.011 ± 0.03

Fig 3 Variation of reducing of membranous nitrate reductase by quinol analogues vs HOQNO concentration The enzyme was reduced with duroquinol (j) or menadiol (d) The nitrate reductase concentration was 0.6 l M Lines were obtained by least-squares fitting.

were distorted at the origin These deviations were due to

the time needed for a homogenous system to be reached

after mixing of the membrane suspension and nitrate

solution during the beginning of measurement for each

experiment Apart from these artifacts the distributions of

the residuals show a good agreement between experimental

data and the plot obtained by fitting

The traces obtained are comparable to these obtained by

Zhao et al [15] These authors found four phases in the

reduction of the cytochrome by menadiol, phases arbitrarily

attributed to particular steps in the scheme that they proposed for the mechanism of reduction Unfortunately, however, this scheme does not agree with the results from steady-state kinetics that we have obtained in a previous study [13] There we showed that the catalytic mechanism implied an intermediate complex incompatible with binding

of the electron donor and allowing it to give its two electrons successively before being released

Spectrophotometric studies with a mutant lacking the highest-potential iron sulphur cluster

Use of a mutant (pVA700-C16) whose nitrate reductase has lost the highest-potential [4Fe-4S] cluster has allowed us to confirm the existence of these two pathways of electron transfer in the enzyme Figure 5 shows reduction of the cytochrome of this mutant by dithionite, menadiol or duroquinol, followed by reoxidation by nitrate The cyto-chrome is unambiguously reduced, regardless of the electron donor On the other hand, although the enzyme reduced

by duroquinol is fully oxidized by nitrate, that reduced by menadiol is not oxidized This result agrees with the conclusions from the steady-state kinetics [13] which showed that in the case of this mutant no menadione oxidase activity was observed even though duroquinone oxidase activity was present The result obtained with dithionite confirms this conclusion; indeed, even through the reduction

is total, the oxidation is only partial, with the fraction of oxidation corresponding to that observed for duroquinol These results indicate therefore that the [Fe–S] clusters

do not have the same role in electron transfer in nitrate reductase The [4Fe-4S] cluster of high-potential would therefore be the indispensable relay for the transfer of electrons when menadiol is the electron donor, and the [3Fe-4S] cluster would be implied in the transfer of electrons when duroquinol is used

Fig 4 Oxidization of membranous nitrate reductase previously reduced

by quinone analogues Absorbance changes observed, at 560 nm, after

mixing enzyme (3.14 l M ) reduced by menadiol (A) or duroquinol (B)

with an equal volume of nitrate (40 m M ) at 37 °C The traces are fitted

to Eqn (1) DAbs ¼ a + b e)ctwhich corresponds to a decreasing

exponential The apparent kinetic constants were 1 s)1for enzyme

reduced by menadiol and 2 s)1for enzyme reduced by duroquinol The

distributions of residuals after fitting data from traces (A) and (B) to

Eqn (1) are shown, respectively, in plots (C) and (D).

Table 2 Comparison of kinetic contants of oxidation by nitrate of nitrate reductase hemes reduced by menadiol or duroqinol Values of kinetic constants were averages of several experiments in different time ranges (0.5, 1, 2 and 5 s) The membranous nitrate reductase concen-tration was in all cases 1.57 l M

[Quinol] (m M ) [KNO 3 ] (m M ) k (s)1) Menadiol

Mean 1.027 ± 0.062 Duroquinol

Mean 1.988 ± 0.094

Steady-state kinetic studies [13] have shown the existence of

two specific binding sites for menadiol and duroquinol This

situation recalls that described for fumarate reductase from

Escherichia coli, where the separation of oxidative and

reductive activities suggests there are two quinol binding

sites and that electron transfer occurs in two one-electron

steps at these sites [26] The presence of these two sites has

been corroborated by crystallographic studies, with two

binding sites termed QPand QDindicating their positions

proximal or distal to the site of fumarate reduction; the use

of the quinol-binding site inhibitor HOQNO shows that the

inhibitor blocks binding of MQH2at the QPsite [27]

Study of the reduction and oxidation of the cytochrome

has brought us to the conclusion that the two hemes do not

have the same role In particular, kinetic results with the

stopped-flow apparatus for the oxidation of the cytochrome

show that there are two distinct reactions, two apparent

kinetic constants differing by a factor of two

Previous studies [13], as well as reduction and oxidation

spectra by nitrate of the cytochrome of a mutant strain

having lost the highest-potential [4Fe-4S] cluster, show that

b clusters do not play the same role in the transfer of

electrons between the cytochrome and the molybdenum

cofactor This [4Fe-4S] cluster is the indispensable relay

when the donor is menadiol The [3Fe-4S] cluster may be the

relay when the donor is duroquinol We can therefore

conclude that the transfer of electrons up to the

molyb-denum cofactor in nitrate reductase follows two distinct

pathways according to the nature of the electron donor,

as illustrated in Fig 6

As the reduction of nitrate to nitrite requires two

electrons, there must necessarily be two successive bindings

of quinone, with transfer of one electron to the hemes, then

to the [Fe-S] cluster, to be finally accumulated at the level of

the molybdenum cofactor to be able to undertake the

catalytic reaction

The location of the hemes has been studied by Rothery

et al [28,29] These authors showed that the low-potential

heme bL, located on the periplasmic side of the membrane,

is associated with a single quinol-binding site However,

the technique used to determine binding sites of quinols,

inhibition by HOQNO (a structural analogue of

menaqui-none), did not allow them to see the binding site of

ubiquinol because HOQNO is not an inhibitor for the

ubiqinones Indeed, cocrystallization of quinol fumarate

reductase with HOQNO shows that HOQNO can inhibit the menaquinol binding site but not the ubiquinol binding site [27] Consequently, the low-potential heme located on the periplasmic side of the membrane appears to be associated with a binding site for menaquinols, but it is highly probable that the high-potential heme bH, located on the cytoplasmic side, was associated with the binding site for ubiquinols This conclusion agrees with structural data that indicate a cavity containing two distinct regions directly adjacent to the two hemes of c [7]

The four [Fe-S] clusters of b are positioned in two pairs due to the fact of their coordination to the polypeptide chain, each high-potential cluster being associated with a low-potential cluster [10] The crystal structure of b shows two structural domains Each domain contains a high-potential [Fe-S] cluster and low-high-potential [Fe-S] cluster, the two being sandwiched between two helices on one side, and

Fig 5 Spectra of membranous nitrate

reduc-tase cytochrome of mutant devoid of the

high-est-potential [4Fe-4S] cluster Reduced spectra

are shown as solid lines and oxidized spectra

as dotted lines The nitrate concentration was

in all cases 0.95 l M

Fig 6 Proposed mechanism for electron transfer in nitrate reductase Menadiol and ubiquinol are symbolized by MQ and UQ, respectively The molybdenum cofactor and the two hemes are denoted CoMo, b L

(low-potential heme) and b H (high-potential heme) This scheme is consistent with the structural data of Bertero et al [ 7].

a b-sheet on the other [7] This structure is consistent with

the idea that the [Fe-S] clusters are coupled in two redox

units, each containing a low-potential and a high-potential

[Fe-S] Thus each structural domain of b may be a transfer

unit for the electron transfer pathway in this enzyme In our

scheme we have integrated these structural data supposing

that the two pairs of [Fe-S] cluster both participate That

recalls that two parallel electron pathways towards the

a [4Fe-4S] cluster and the molybdenum cofactor with

involvement of high-potential [Fe-S] had been suggested,

supposing that the low-potential clusters play no redox role

[17] Note that HOQNO and stigmatellin inhibit

nitrate-dependent heme reoxidation [28], suggesting the presence of

a second dissociable Q-site (the Qnrsite) between heme bH

and the [3Fe-4S] cluster [29] Likewise our results are not at

variance with kinetic data for reduction of the enzyme by

menadiol [15], suggesting the existence of two menadiol

binding sites in the enzyme, one with higher affinity than the

other, as well as inhibition data indicating the possibility of

more than one menaquinol binding site in nitrate reductase

[15] The fact that nitrate is able to oxidize heme bHbut not

heme bLin the presence of an excess of quinol and HOQNO

[30] is consistent with our results showing inhibition by

HOQNO to the extent of reduction by menadiol and not

by duroquinol, and thus inhibition of duroquinone oxidase

activity

The existence of these two pathways of electron transfer

may appear surprising, but nitrate reductase is one of the

rare enzymes of quinone-oxidase type that can accept both

menaquinol and ubiquinol as electron donor according to

conditions of growth

Acknowledgements

We are indebted to Dr F Blasco for his help in the construction of

mutant strains, to Dr G Giordano for preparation of enzymes and to

Dr J Pommier for performing the rocket assays The authors express

their gratitude to Dr Wolfgang Nitschke (BIP, CNRS, Marseille) to

have allowed us to use a stopped-flow apparatus We thank Dr

A Cornish-Bowden for critical reading and correcting of the manuscript.

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