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J. Biol. Chem., Vol. 277, Issue 25, 22402-22406, June 21, 2002

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Nitric Oxide Reacts with the Single-electron Reduced Active Site of Cytochrome c Oxidase^*

Alessandro Giuffrè, Maria Cecilia Barone, Maurizio Brunori, Emilio D'Itri, Bernd Ludwig^§, Francesco Malatesta^¶, Hans-Werner Müller^§, and Paolo Sarti

From the Department of Biochemical Sciences and CNR Institute of Molecular Biology and Pathology, University of Rome "La Sapienza," I-00185 Rome, Italy, the ^§ Institute of Biochemistry, Molecular Genetics, University of Frankfurt, Biozentrum, D-60439 Frankfurt, Germany, and the ^¶ Department of Pure and Applied Biology, University of L'Aquila, I-67100 L'Aquila, Italy

Received for publication, February 14, 2002, and in revised form, April 3, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa₃) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a₃ reduction, which is extremely slow in the K354M mutant (k₁ = 0.09 ± 0.01 s¹; k₂ = 0.005 ± 0.001 s¹) but much faster in the D124N aa₃ (k₁ = 21 ± 6 s¹; k₂ = 2.2 ± 0.5 s¹). NO causes a very large increase (>100-fold) in the rate constant of heme a₃ reduction in the K354M mutant but only a ~5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O₂ when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a₃ reduction is ~100-fold faster than the extremely low turnover rate, we conclude that, contrary to O₂, NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H⁺-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytochrome c oxidase (CcOX)¹ contains a bimetallic active site (heme a₃-Cu_B) where O₂ is reduced to H₂O. This exergonic reaction is coupled to an active translocation of protons, generating a proton-motive force used for ATP synthesis (see Refs. 1 and 2 for reviews). Complete reduction of the heme a₃-Cu_B center, a prerequisite for the reaction with O₂, occurs via intramolecular electron transfer from heme a, which in turn is reduced by Cu_A, the metal center accepting electrons from cytochrome c. Protons (both scalar and vectorial) are made available in situ via two putative H⁺-conducting pathways, identified in the crystallographic structure (3, 4). These pathways, called K and D from the residues Lys-354² and Asp-124 of subunit I, play different roles in the mechanism, as extensively investigated by site-directed mutagenesis (see Refs. 1, 2, and 5 for reviews).

The catalytic cycle of cytochrome c oxidase can be divided into a reductive and an oxidative part. In the reductive part, two electrons are sequentially transferred to the fully oxidized heme a₃-Cu_B center called O, yielding the two-electron reduced site R via a single-electron reduced intermediate E. In the oxidative part, upon reaction with O₂, R restores the fully oxidized enzyme O, by populating the O₂ intermediates P and F (depending on the redox state of heme a, two different P intermediates are formed, called P_M and P_R). The idea that the O  R process is the rate-determining step in the overall catalytic cycle is gaining further support (6-8).

Mutation of Lys-354 to M within the K pathway yields a virtually inactive enzyme, as shown for the Rhodobacter sphaeroides aa₃ (9-11), the Escherichia coli bo₃ (12), and the Paracoccus denitrificans aa₃ (5). This mutation affects primarily, but not exclusively (see Ref. 13), the reductive part of the catalytic cycle (9, 11, 14); in the absence of O₂ and with a large excess of reductant, heme a₃ is reduced at an extremely low rate (time scale of several minutes) as compared with the wild type (time scale of tens of milliseconds). This reduction block is presumably due to an impaired H⁺ transfer in the K354M mutant, consistent with the loss of the millisecond phase in laser-triggered reverse electron transfer experiments observed with the analogous mutant of the R. sphaeroides enzyme (15). Recently, two groups (16-18) reported time-resolved electrometric measurements on liposome-reconstituted mutants of the P. denitrificans CcOX by laser excitation of ruthenium(II) bispyridyl. According to Ruitenberg et al. (16), injection of a single electron into the oxidized enzyme is coupled to an H⁺ transfer through the K pathway, linked to reduction of heme a. In contrast, Verkhovsky et al. (18) proposed that an H⁺ uptake through the K pathway controls the single-electron reduction of heme a₃-Cu_B (O  E), impaired in the K354M mutant, whereas the formation of the two-electron reduced active site (E  R) would be coupled to an H⁺ uptake through the D pathway, as deduced from data on the inactive D124N mutant (17). The first of the two protons taken upon reduction of the active site has been proposed to charge-compensate the reduction of Cu_B in the single-electron reduced active site via protonation of a putative OH bound to this metal in the oxidized state (1, 17).

The effect of the K354M mutation is drastic in the reductive part of the catalytic cycle but much smaller in the oxidative part. As assessed by the flow-flash technique using the R. sphaeroides CcOX analogous to the K354M mutant, the fully reduced enzyme exposed to O₂ becomes fully oxidized within ~5 ms (15), although without the formation of P_R (13). The loss of oxidase activity associated to the K354M mutation has been therefore assigned to the extremely slow formation of R (9, 11, 14), which is a prerequisite for the reaction with O₂.

Differently from O₂, NO has been suggested to bind not only to R (19) but also to a single-electron reduced intermediate E (20, 21). This hypothesis, raised to account for the very low apparent K_i for NO inhibition, although consistent with computer simulations (20, 21), is not yet supported by direct experimental evidence. In this report, we provide evidence for the reaction of NO with E by studying the kinetics of reduction of the K354M and D124N mutants of P. denitrificans CcOX in the presence of NO.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dodecyl--D-maltoside was purchased from Biomol (Hamburg, Germany); ascorbate, glucose oxidase, and catalase were purchased from Sigma; and ruthenium(III) hexamine was purchased from Aldrich. Stock solutions of NO (Air Liquide, Paris, France) were prepared by equilibrating degassed water with the pure gas at 1 atm ([NO] = 2 mM at 20 °C).

The K354M and D124N mutants of cytochrome c oxidase from P. denitrificans were purified according to Ref. 22 and stored at 80 °C. Before use, the enzymes were equilibrated by dialysis (at 4 °C for at least 5 h) with the buffer used in the experiments (100 mM K⁺/phosphate, pH 7.0, + 0.1% dodecyl maltoside or 35 mM K⁺/phosphate, pH 7.0, + 50 mM KCl + 0.1% dodecyl maltoside). Cytochrome oxidase concentration is expressed in terms of functional units (aa₃) using the extinction coefficient _{red-ox, 444} = 156 mM¹ cm¹.

Stopped-flow experiments were carried out with a DX.17MV Applied Photophysics instrument equipped with a diode array (Leatherhead, UK). The mixing apparatus allows rapid mixing of equal volumes of solutions either in a simple or a sequential mode; in the latter mode, two solutions are premixed, and after a preset delay, they are mixed again with another solution. The instrument has a 1-cm light path and can acquire absorption spectra with an acquisition time of 2.5 ms. In a typical experiment, ascorbate (80 mM) and ruthenium hexamine (4 mM) are premixed with N₂-equilibrated buffer (with or without NO), and the resulting solution is mixed after a 100-ms delay with degassed oxidized CcOX at 20 °C. This protocol prevents prolonged incubation of reductants with NO. Contaminant oxygen was scavenged with glucose and catalytic amounts of glucose oxidase and catalase. After the second mixing, absorption spectra were collected as a function of time according to a logarithmic scale. Data analysis was carried out using the software MATLAB (The MathWorks, South Natick, MA). Spectral smoothing and deconvolution were performed by using the singular value decomposition (SVD) algorithm according to Henry and Hofrichter (23) or by the pseudoinverse algorithm.

The stoichiometry of NO binding to the K354M CcOX has been measured according to Stubauer et al. (24) by using a NO-selective Clark-type electrode (ISO-NO, World Precision Instruments). The electrode is calibrated using aliquots of NO-saturated water added to the degassed buffer and, after the addition of CcOX, the concentration of NO in solution is monitored.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present investigation, we studied by stopped-flow spectrophotometry the kinetics of reduction of the K354M and the D124N mutants of the P. denitrificans CcOX both in the presence and in the absence of NO. As shown in Fig. 1, the K354M mutation yields a dramatic decrease in the rate of heme a₃ reduction, consistent with the literature (9, 11, 14, 17). Upon mixing anaerobically oxidized K354M CcOX with a large excess of ascorbate and ruthenium hexamine, heme a reduction is very fast (~7 ms), whereas reduction of heme a₃ is extremely slow (>500 s, Fig. 1A). SVD analysis of the latter process shows a single significant optical component (corresponding to the reduced minus oxidized heme a₃ spectrum), displaying a biphasic time course (Fig. 1C). Best fit of the time course yields k₁ = 0.09 ± 0.01 s¹ and k₂ = 0.005 ± 0.001 s¹ for the two phases, accounting for ~30 and 70% of the total amplitude, respectively. We cannot exclude that the intrinsic reduction rate might be even slower than observed given that over the very long time scale explored (500 s), the high intensity light beam of the diode array instrument causes partial enzyme reduction even in the absence of reductants (data not shown). In agreement with others (5, 9, 11), we conclude that the very slow heme a₃ reduction may account for the extremely slow turnover observed with O₂ (~0.02 mol of O₂/mol of CcOX × s at 170 µM cytochrome c)

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Reduction of the K354M aa3 and the effect of NO. The oxidized K354M mutant of the P. denitrificans cytochrome oxidase is anaerobically mixed with reductants in the absence (A) and in the presence of NO (B). Final concentrations: [ascorbate] = 20 mM; [ruthenium hexamine] = 1 mM; [CcOX] = 2.2 µM aa3. T = 20 °C. As shown in A and B, the enzyme, initially in the oxidized state, is rapidly (7 ms) reduced in its heme a moiety both in the absence and in the presence of NO. Afterward, in the absence of NO (A), complete reduction is achieved very slowly (500 s), whereas in the presence of NO (B), the formation of reduced NO-bound heme a3 is complete at 12 s. The solid spectra in both panels are end-point species and correspond to the reduced enzyme either uncomplexed (A) or NO-bound (B). C, time courses of heme a3 reduction as obtained by SVD analysis. In the absence of NO, heme a3 is reduced very slowly with rate constants of k1 = 0.09 s1 (30% total amplitude) and k2 = 0.005 s1 (70% total amplitude). In the presence of NO, heme a3 reduction is much faster with rate constants of k1 = 8.9 s1 (50% total amplitude) and k2 = 0.58 s1 (50% total amplitude).

A different scenario is observed when the reduction of the K354M CcOX is carried out in the presence of NO (Fig. 1B). In these experiments, the stopped-flow apparatus was used in the sequential mixing mode to prevent the prolonged incubation of NO with the reductants in the stopped-flow syringe to avoid NO loss (see "Experimental Procedures"). At 500 µM NO (concentration after mixing), heme a reduction is again complete within a few milliseconds. In this case, however, the end point species (i.e. the fully reduced enzyme with NO bound to heme a₃) is already populated after about 10 s, indicating a much faster internal electron transfer in the presence of NO. SVD analysis of the absorption spectra collected from 7 ms up to 10 s reveals only a single optical component corresponding to the [heme a-NO]-[heme a] difference spectrum, indicating that NO binding is rate-limited by (and thus, apparently synchronous with) heme a₃ reduction. Analysis of the time course in Fig. 1C shows that the time course of heme a-NO formation is biphasic with rate constants of k₁ = ~8.9 s¹ and k₂ = ~0.6 s¹, the two phases having similar amplitude. Thus, NO seems not to interfere with heme a reduction (very fast both with and without NO) but clearly drives heme a₃ reduction, which occurs in the presence of NO at least 100-fold faster than in its absence (Fig. 1C).

The experiments reported above were extended to the D124N mutant (Fig. 2). In the latter mutant, similarly to the K354M mutant, the reduction of heme a is fast both in the presence and in the absence of NO, being complete within a few milliseconds after mixing with reductant (Fig. 2, A and B). In agreement with the literature (17), the enzyme is completely reduced within a few seconds even in the absence of NO (compare Figs. 1A and 2A), and thus, heme a₃ reduction is much faster than in the case of the K354M mutant. Also, for the D124N mutant, heme a₃ reduction is biphasic with k₁ = 21 ± 6 s¹ and k₂ = 2.2 ± 0.5 s¹ (relative amplitudes ~70 and 30%, respectively). Complete reduction of the D124N mutant is accelerated in the presence of NO (Fig. 2B) and, for instance, at 500 µM NO, the formation of the heme a-NO complex proceeds at k₁ = 74 ± 11 s¹ and k₂ = 3.6 ± 1.6 s¹ (relative amplitudes ~60 and 40%, respectively). Thus, we conclude that in both mutants, the addition of NO increases the rate of internal electron transfer; however, this increase corresponds to a factor of ~5 for the D124N mutant and to >100-fold in the K354M mutant. This result is better visualized in Fig. 3, in which the two observed rate constants of heme a₃ reduction for both mutants are reported at different NO concentrations (from 0 to 500 µM). The data show that internal electron transfer in the D124N mutant is faster than in the K354M mutant. Moreover, in both mutants, the two rate constants (relative to the fast and the slow kinetic phases) depend on the NO concentration. Although the dependence is much less pronounced in the D124N mutant, all rate constants become essentially independent of [NO], reaching plateau values at k₁ = 13 ± 6 s¹ and k₂ = 0.7 ± 0.3 s¹ in the K354M mutant and k₁ = 110 ± 16 s¹ and k₂ = 3.5 ± 1.5 s¹ in the D124N mutant. All asymptotic values are independent of reductant concentration, as assessed by experiments at 500 µM NO and variable ruthenium hexamine concentration; at ruthenium hexamine concentrations above 1 mM (after mixing), the observed rate constants were independent of reductant concentration.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Reduction of the D124N aa3 and the effect of NO. The oxidized D124N mutant of the P. denitrificans cytochrome oxidase is anaerobically mixed with reductants in the absence (A) and in the presence of NO (B). Experimental conditions are as described in the legend for Fig. 1. A and B, similar to the K354M aa3, heme a reduction is complete within a few milliseconds, independently of NO. Afterward, complete reduction of the enzyme is achieved within a few seconds in the absence of NO (A) and on an even shorter time scale in the presence of NO (B). C, time courses of heme a3 reduction as obtained by the pseudoinverse analysis. In the absence of NO, heme a3 is reduced with rate constants of k1 = 15 s1 (60% total amplitude) and k2 = 2.7 s1 (40% total amplitude). At 500 µM NO, heme a3 reduction is faster and proceeds with rate constants of k1 = 78 s1 (60% total amplitude) and k2 = 5.1 s1 (40% total amplitude).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of NO concentration. Rate constants of heme a3 reduction, relative to the fast (top) and the slow (bottom) phases, were measured at varying NO concentrations. Experimental conditions were as described in the legend for Fig. 1. Under all conditions, internal electron transfer in the D124N mutant is faster than in the K354M mutant. Both rate constants depend on the NO concentration, although the dependence is much less pronounced in the D124N mutant.

It is worth noticing that the faster heme a₃ reduction observed with both mutants in the presence of NO is not due to direct reduction of the oxidized binuclear center O by NO. As demonstrated with beef heart CcOX and confirmed with the P. denitrificans wild-type CcOX, the reaction of NO with O occurs rapidly with the chloride-free enzyme, yielding nitrite-bound heme a₃ and reduced heme a (25), but it is prevented with the chloride-bound oxidase (26). Spectrophotometrically, we did not detect a reaction after mixing both oxidized mutants with NO (1 mM after mixing, not shown). Further in this respect, the reactivity of NO with the oxidized K354M oxidase was probed by amperometry by using a NO-selective Clark-type electrode (24). If the oxidized enzyme is anaerobically added to a degassed NO-containing solution, a reaction would be detected as a decrease in the NO concentration. Upon the addition of oxidized K354M CcOX (0.4 µM) to a solution containing NO (1 µM), only a small decrease in the NO concentration (~0.2 mol of NO/mol of oxidase) was detected; in contrast, a stoichiometric (1:1) NO binding was observed after the addition of the fully reduced enzyme, as shown for the mammalian CcOX (24). We therefore conclude that both the P. denitrificans mutants tested in this study are in the chloride-bound state, due to the presence of chloride in the buffers used during both the purification and the experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NO is a very efficient, yet reversible, inhibitor of cytochrome c oxidase activity (27, 28), leading to the proposal that it may act as a physiological modulator of cell respiration (29). Since both NO and O₂ react with the fully reduced heme a₃-Cu_B center R with high affinity and similar rates (19), the small inhibition constant K_i determined with mitochondrial CcOX in turnover (K_i = 270 nM NO at [O₂] = 140 µM, (27)) was somewhat puzzling. To account for this observation, it was proposed that NO can react with a single-electron reduced active site E (20-21), which is known to be unreactive toward O₂. Such a hypothesis is consistent with computer simulations (20-21) but has never been demonstrated experimentally. The kinetics of reduction of the K354M mutant of P. denitrificans in the presence of NO, reported above, provides evidence that this hypothesis is correct.

The K354M mutation is associated with the loss of oxidase activity (5, 9, 11), although this mutant in the fully reduced state (R) is very quickly (~5 ms) oxidized by O₂, as reported for the R. sphaeroides CcOX (15, but see also Ref. 13). The same mutation has a dramatic effect on the reductive part of the catalytic cycle, and the extremely low rate of reduction of heme a₃ was correlated to the marginal turnover rate (9, 11, 14). It was therefore assumed that in this mutant, the turnover with O₂ is rate-limited by the extremely slow formation of R, which is an obligatory intermediate in the catalytic cycle. This is consistent with the widely accepted idea that O₂ can react exclusively with the two-electron reduced heme a₃-Cu_B site, like CO.

The novel result reported in this study on the K354M mutant of P. denitrificans CcOX is that, in the presence of NO, the reduction of heme a₃ occurs at a rate much faster (>100-fold) than in its absence and much faster than the extremely low turnover rate of this mutant with O₂ (Fig. 1). This effect depends on NO concentration and is maximal at [NO] >100 µM (Fig. 3), at which the reduction of heme a₃ proceeds at rates (k₁ = 13 ± 6 s¹; k₂ = 0.7 ± 0.3 s¹) both remarkably larger than the turnover rate (~0.02 mol of O₂/mol of CcOX × s at [O₂] > 250 µM). This finding is diagnostic of a different reactivity of O₂ and NO with CcOX. It is indeed difficult to account for this result, assuming that NO, similarly to O₂, can react exclusively with R, which combines with very high affinity and second order rate constants with both ligands. If this were the case, the formation of the reduced NO-bound heme a₃ would be much slower, being rate-limited by the formation of R, which in turn accounts for the extremely low oxidase activity. Therefore this result implies that NO can react not only with the two-electron reduced heme a₃-Cu_B site R (19) but also with the single-electron reduced site E, whose occurrence in this mutant was already documented (11). We do not have a valid explanation for the observed heterogeneity in the reduction of heme a₃, but we notice that a similar biphasic behavior was reported also for the beef heart enzyme (7, 8).

Working with beef CcOX, it was shown that the reaction of NO with Cu_B in the oxidized binuclear site (O) occurs rapidly only with the chloride-free enzyme, leading to reduced heme a and nitrite-bound oxidized heme a₃ (25) but is prevented by the binding of chloride (26). This behavior was reproduced with the P. denitrificans wild-type enzyme. On mixing either of the mutants in the oxidized state with a large excess of NO, we did not observe spectrophotometrically either heme a reduction or nitrite formation. Moreover, we observed by amperometry only a small reaction between NO and oxidized K354M (similar to that generally detected even with the beef heart enzyme in the chloride-bound form, see Ref. 26). We therefore conclude that the two mutants employed in this study are in the Cl-bound form since chloride is present in the buffers used during the purification and the experiments. This further implies that the enhanced reduction of heme a₃ observed in the presence of NO cannot be assigned to the direct reaction of NO with O.

It is noteworthy that the dependence on [NO] reported in Fig. 3 is consistent with the idea that the K354M mutation impairs the O  E electron transfer step (17, 18). Assuming that NO binds to heme a in the E and the R states with very high affinity and second order rate constants, one may expect that NO should efficiently drive thermodynamically the reduction of heme a₃ in the O  E step. On this basis, the NO concentration dependence of the apparent rate constants measured for the K354M mutant (Fig. 3) seems diagnostic of a relatively slow forward electron transfer (maximal value of 13 ± 6 s¹) as compared with an unusually fast reverse electron transfer (heme a₃/Cu_B  heme a) caused by the mutation. In this context, it is interesting to notice that the D124N mutant shows a kinetic behavior remarkably different from the one displayed by the K354M mutant. In the D124N mutant, (i) heme a₃ reduction in the absence of NO is much faster than in the K354M mutant, in agreement with Wikström et al. (17), and (ii) NO increases this rate at most by a factor of ~5 (ratio of the rate constant at saturating [NO] over the value measured in the absence of NO), i.e. much less than the over 100-fold increase observed with the K354M mutant. These results are fully consistent with the hypothesis that the K pathway, but not the D pathway, controls the first electron transferred to the oxidized heme a₃-Cu_B site (16-18).

The analogous K354M mutant of the R. sphaeroides CcOX has been reported to display cytochrome c-peroxidase activity with a K_m value of ~50 mM H₂O₂ and a V_max value of ~25 s¹ (14, 30). The maximal turnover with H₂O₂ was much faster than the turnover with O₂, and this result was interpreted as an evidence that H₂O₂ reacts with O, yielding the intermediate P, i.e. bypassing the whole reductive part of the catalytic cycle (14). Later on, it was proposed that H₂O₂ might react with E in the K354M mutant, yielding directly the F intermediate, thus bypassing the formation of both R and P (2, 31). We wish to point out that according to our results, the latter interpretation has to be favored, and in this respect, H₂O₂ and NO behave similarly since they are both assumed to react with E. We notice that the maximal turnover number for the peroxidase activity (25 s¹, see Ref. 14) is not inconsistent with the faster rate constant for the O  E process that we estimate from our data on the K354M mutant at saturating [NO] (13 ± 6 s¹; Fig. 3, top panel). In other words, we propose that in the K354M mutant, the rate constant of heme a₃ reduction measured at saturating concentrations of NO and ruthenium hexamine is the forward rate constant for the single-electron reduction of the heme a₃-Cu_B site, which is also rate-limiting the cytochrome c-peroxidase activity at saturating [H₂O₂].

In conclusion, our results provide direct evidence that NO, differently from O₂, can react with a single-electron reduced heme a₃-Cu_B in CcOX. This finding validates the original hypothesis (20, 21) raised to account for the finding that NO is a potent inhibitor of CcOX (27, 28).

	ACKNOWLEDGEMENT

We thank Dr. Viktoria Drosou for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported in part by a Vigoni Program grant from the Conferenza dei Rettori delle Università Italiane and Deutscher Akademischer Austauschdienst (to M. B., F. M., and B. L.), by a Programma di Ricerca Scientifica Interuniversitario Nazionale "Bioenergetica: aspetti genetici, biochimici e fisiopatologici" from the Ministero dell'Istruzione, dell'Università e della Ricerca of Italy (to P. S. and F. M.), and by Grant SFB 472 from the Deutsche Forschungsgemeinschaft (to B. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Instituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche, c/o Dipartimento di Scienze Biochimiche "A. Rossi. Fanelli", Università di Roma "La Sapienza," Piazzale Aldo Moro 5, I-00185 Roma, Italia. Tel.: 39-06-4450291; Fax: 39-06-4440062; E-mail: alessandro.giuffre@uniroma1.it.

Published, JBC Papers in Press, April 11, 2002, DOI 10.1074/jbc.M201514200

² The amino acid numbering is based on the P. denitrificans cytochrome c oxidase sequence.

	ABBREVIATIONS

The abbreviations used are: CcOX, cytochrome c oxidase; SVD, singular value decomposition; O, enzyme with oxidized heme a₃-Cu_B site; E, enzyme with a single-electron reduced heme a₃-Cu_B; R, enzyme with a two-electron reduced heme a₃-Cu_B..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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