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A Common Mechanism for the Interaction of Nitric Oxide with the Oxidized Binuclear Centre and Oxygen Intermediates of Cytochromec Oxidase*

  • Jaume Torres
    Affiliations
    Department of Biological Sciences, University of Essex, Wivenhoe Park, CO4 3SQ Colchester, Essex, United Kingdom
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  • Chris E. Cooper
    Affiliations
    Department of Biological Sciences, University of Essex, Wivenhoe Park, CO4 3SQ Colchester, Essex, United Kingdom
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  • Michael T. Wilson
    Correspondence
    To whom correspondence should be addressed. Tel.: 44-1206-872538; Fax: 44-1206-872592
    Affiliations
    Department of Biological Sciences, University of Essex, Wivenhoe Park, CO4 3SQ Colchester, Essex, United Kingdom
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  • Author Footnotes
    * This work was supported by the Wellcome Trust, the Royal Society, the Spanish government, and the Biotechnology and Biological Science Research Council, UK.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.
Open AccessPublished:April 10, 1998DOI:https://doi.org/10.1074/jbc.273.15.8756
      The reactions of nitric oxide (NO) with fully oxidized cytochrome c oxidase (O) and the intermediates P and F have been investigated by optical spectroscopy, using both static and kinetic methods. The reaction of NO with O leads to a rapid (∼100 s−1) electron ejection from the binuclear center to cytochrome a and CuA. The reaction with the intermediates P and F leads to the depletion of these species in slower reactions, yielding the fully oxidized enzyme. The fastest optical change, however, takes place within the dead time of the stopped-flow apparatus (∼1 ms), and corresponds to the formation of the F intermediate (580 nm) upon reaction of NO with a species that we postulate is at the peroxide oxidation level. This species can be formulated as either Fe5+ = O CuB2+or Fe4+ = O CuB3+, and it is spectrally distinct from the P intermediate (607 nm). All of these reactions have been rationalized through a mechanism in which NO reacts with CuB2+, generating the nitrosonium species CuB1+ NO+, which upon hydration yields nitrous acid and CuB1+. This is followed by redox equilibration of CuB with Fea/CuA or Fea3 (in which Fea and Fea3 are the iron centers of cytochromes a and a3, respectively). In agreement with this hypothesis, our results indicate that nitrite is rapidly formed within the binuclear center following the addition of NO to the three species tested (O, P, and F). This work suggests that nitrosylation at CuB2+ instead of at Fea32+ is a key event in the fast inhibition of cytochrome c oxidase by NO.
      Cytochrome c oxidase (ferrocytochromec oxidoreductase, EC 1.9.3.1), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the reduction of molecular oxygen to water (
      • Babcock G.T.
      • Wikström M.
      ). This process is coupled to proton translocation across the inner membrane. The enzyme contains four redox-active centers. Electron entry from cytochrome c, the natural substrate, occurs via a diatomic copper center, CuA. After rapid equilibrium with Fea,
      The abbreviations used are: Fea and Fea3, iron centers of cytochromesa and a3, respectively; CcO, cytochrome c oxidase; NO, nitric oxide; O, fully oxidized cytochrome c oxidase formed after reduction and reoxidation of the enzyme; Ob, oxidized binuclear center of CcO; P and F, oxygen intermediates of CcO at an oxidation state two and three electrons more reduced, respectively, than the oxidized binuclear center; PM, species formed immediately after CO-photodissociation from mixed valence-CO in the presence of oxygen; PR, species formed immediately after CO-photodissociation from the fully reduced CO complex in the presence of oxygen.
      1The abbreviations used are: Fea and Fea3, iron centers of cytochromesa and a3, respectively; CcO, cytochrome c oxidase; NO, nitric oxide; O, fully oxidized cytochrome c oxidase formed after reduction and reoxidation of the enzyme; Ob, oxidized binuclear center of CcO; P and F, oxygen intermediates of CcO at an oxidation state two and three electrons more reduced, respectively, than the oxidized binuclear center; PM, species formed immediately after CO-photodissociation from mixed valence-CO in the presence of oxygen; PR, species formed immediately after CO-photodissociation from the fully reduced CO complex in the presence of oxygen.
      the electron is transferred to CuB and Fea3, which together constitute the binuclear center, where oxygen is reduced.
      Although oxygen binds with low affinity (103m−1) (
      • Verkhovsky M.I.
      • Morgan J.E.
      • Wikström M.
      ) to reduced Fea3, rapid electron transfer from the two reduced metals comprising the binuclear center to molecular oxygen ensures that oxygen remains bound as a peroxy species. In this way, oxygen is kinetically trapped and further reduction can take place (
      • Chance B.
      • Saronio C.
      • Leigh Jr., J.S.
      ,
      • Verkhovsky M.I.
      • Morgan J.E.
      • Puustinen A.
      • Wikström M.
      ). The successive steps leading to the formation of water have been studied by different spectroscopic techniques (see Ref.
      • Ferguson-Miller S.
      • Babcock G.T.
      for review). The spectral signatures of two of these intermediates, which exhibit bands at 607 and 580 nm in the difference spectrum with respect to the oxidized enzyme, were first reported by Wikström (
      • Wikström M.
      ), by reversing the electron transfer reaction, and further characterized by Wikström and Morgan (
      • Wikström M.
      • Morgan J.E.
      ). These authors assigned the spectral signatures at 607 and 580 nm to a ferric peroxy (P) and ferryl oxo (F) species, respectively. These assignments, however, have been challenged by a number of authors (
      • Weng L.C.
      • Baker G.M.
      ,
      • Fabian M.
      • Palmer G.
      ,
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S.
      • Appelman E.H.
      • Kitagawa T.
      ). For example, Resonance Raman results obtained by Proshlyakovet al. (
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S.
      • Appelman E.H.
      • Kitagawa T.
      ) suggest that the 607 nm band originates from an oxoferryl structure. On the other hand, the same authors have been unable to identify the putative peroxy species in their system in turnover sustained by H2O2 (
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S
      • Kitagawa T.
      ). However, irrespective of the assignments, there seems to exist a general agreement that compound F (580 nm) is a ferryl oxo species and that it is one electron more reduced than compound P (607 nm) (
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S
      • Kitagawa T.
      ,
      • Verkhovsky M.I.
      • Morgan J.E.
      • Wikström M.
      ).
      One of the ways to solve the problem of the identity of the P intermediate could perhaps be through the use of a suitable probe. The possibility that nitric oxide (NO), a powerful reversible inhibitor of cytochrome c oxidase (
      • Cleeter M.W.J.
      • Cooper J.M.
      • Darley-Usmar V.M.
      • Moncada S.
      • Schapira A.H.V.
      ,
      • Brown G.C.
      • Cooper C.E.
      ,
      • Torres J.
      • Darley-Usmar V.M.
      • Wilson M.T.
      ), can be used as a probe for the binuclear center has been suggested by recent results using enzyme in slow turnover (
      • Torres J.
      • Cooper C.E.
      • Wilson M.T.
      ,
      • Wilson M.T.
      • Torres J.
      • Cooper C.E.
      • Sharpe M.
      ,
      • Cooper C.E.
      • Torres J.
      • Sharpe M.A.
      • Wilson M.T.
      ). In these experiments, an electron was ejected from the binuclear center, partially reducing cytochromea. In addition, depletion of the intermediates P and F was also observed on mixing with NO (
      • Torres J.
      • Cooper C.E.
      • Wilson M.T.
      ,
      • Wilson M.T.
      • Torres J.
      • Cooper C.E.
      • Sharpe M.
      ). As suggested in these papers, these reactions could be mediated via reaction of NO with CuB, as it has been shown that in addition to binding to reduced Fea3, NO binds to both CuB1+ and CuB2+, albeit with different affinities (
      • Stevens T.H.
      • Brudwig G.W.
      • Bocian D.F.
      • Chan S.I.
      ,
      • Brudvig G.W.
      • Stevens T.H.
      • Chan S.I.
      ,
      • Zhao X.J.
      • Sampath V.
      • Caughey W.S.
      ). On the other hand, we have suggested previously (
      • Torres J.
      • Darley-Usmar V.M.
      • Wilson M.T.
      ) that binding of NO to CuB1+ could be a key to understanding the mechanism of inhibition of CcO by NO, although this hypothesis was based solely on steady-state kinetic considerations.
      A mechanism describing the interaction of NO with CuB, leading to the partial reduction of cytochrome a, could be the reverse of that postulated for the reduction of nitrite to NO by non-heme nitrite reductases, which contain only Cu as a metal (see Ref.
      • Averill B.A.
      for review). In this mechanism, depicted in SchemeFS1 (solid arrows), Cu1+ of nitrite reductase (A) binds nitrite, forming a complex (B), which after abstraction of one oxygen atom gives an electrophilic nitrosonium (C). This species is analogous to the nitrosonium (Fe2+ NO+), detected by Fourier transform infrared spectroscopy in the heme-containing cytochrome cd1 nitrite reductase ofPseudomonas stutzeri, obtained by incubating the oxidized enzyme with NO (
      • Yang Y.
      • Averill B.A.
      ). The donation of one electron from the metal gives Cu2+ NO (D), which then dissociates as NO and oxidized Cu (E).
      We suggest that in CcO, NO interacts with the oxidized enzyme at the copper present in the binuclear center, CuB2+, forming a complex analogous to that shown at point D in Scheme FS1. This eventually yields nitrite and CuB1+ (Scheme FS1, dotted arrows, E through A). As depicted in SchemeFS2, the reduced CuB could be reoxidized by equilibration with the other redox centers,i.e. Fea, CuA, and Fea3. In those cases in which oxygen intermediates are present in the binuclear center, an obvious possible outcome of the internal electron transfer from CuB to Fea3 would be the transition of the oxygen intermediate to the next intermediate in the catalytic cycle, i.e. P → F or F → Ob(Ob represents the oxidized binuclear center of CcO, Fea33+CuB2+). Alternatively, CuB1+ could bind NO, forming a relatively stable complex, Cu1+ NO. The formation of this complex could ultimately be responsible for the inhibition of the enzyme.
      In this study, we have investigated in detail these reactions with a view to test the hypotheses formulated in Schemes FS1 and FS2. We provide evidence for the mechanisms depicted in these schemes, which are able to explain many of the features encountered in the reaction of NO with CcO.

      MATERIALS AND METHODS

      Cytochrome c oxidase was prepared by the method of Soulimane and Buse (
      • Soulimane T.
      • Buse G.
      ), which yields highly active enzyme (maximal turnover number, 600 s−1). The buffer used throughout was 0.1 m HEPES, 0.5% Tween 80, pH 7.4. Because of the sluggish reactivity of the binuclear center of this enzyme as preparation with CO, H2O2 (used to form P and F, respectively), and NO, the enzyme was “pulsed” by reduction and reoxidation. This was necessary both to prepare the intermediates P and F and to investigate the reactivity of the oxidized enzyme with NO.

      Pulsed Oxidized Enzyme O

      CcO (∼45 μm) was fully reduced by incubation with 1 mmsodium dithionite for 2 h at 4 °C. Then, the enzyme was reoxidized by passage through a Sephadex G-25 column equilibrated with the same buffer but containing no dithionite. To ensure that the enzyme was fully reoxidized, the column was loaded, immediately before the addition of the enzyme, with a band of 20 mm potassium ferricyanide. Formation of peroxide upon reoxidation was avoided by addition of 50 μl of 40 mm catalase to the reduced enzyme prior loading onto the column. Following this procedure, the maximum of the Soret band corresponding to the pulsed enzyme was at 423 nm, identical to the maximum observed in the oxidized enzyme as prepared. Formation of oxygen intermediates of the enzyme (e.g.peroxy), due to either incomplete reoxidation or the formation of peroxide, would induce a red shift of the Soret band. The absence of a significant shift in this band after pulsing is indicative of the absence of such oxygen intermediates. The pulsed oxidized enzyme (O) thus obtained was used to prepare the intermediates P and F.

      Compounds P and F

      Compound F was formed by incubating O (see above) in 1 mm H2O2, and the concentration of F was determined spectroscopically using Δε580–630 = 5,500 m−1cm−1 (
      • Wikström M.
      • Morgan J.E.
      ) relative to O. Compound P was obtained by two different methods. In the first method, P was formed when CO gas was bubbled for a short time (∼10 s) through a solution containing typically ∼4 μm O (see above) as described previously (
      • Nicholls P.
      • Chanady G.A.
      ). After about 10 min, formation of P reached a steady state, with conversion of typically ∼50% of the enzyme to this form. In the second method, the oxidized enzyme (pulsing was not necessary) was incubated with CO overnight at 4 °C to obtain the mixed valence-CO complex. After briefly degassing the solution, the sample was exposed to an intense flash of white light in the presence of oxygen. The amount of compound P formed using this method was typically ∼80% of the total enzyme, as determined using the extinction coefficient Δε607–630 = 11,000 m−1cm−1 (
      • Wikström M.
      • Morgan J.E.
      ) relative to O.

      Static Experiments

      In the static experiments, an aliquot of a solution of NO (50 μl of 2 mm NO) was added to 1.5 ml of a solution containing compound P (607 nm), F (580 nm), or O (fully oxidized enzyme). A difference spectrum relative to O was recorded immediately in a Cary 5E UV-Vis-NIR spectrophotometer. The amount of cytochrome a reduced was calculated as follows: Δε605 (reduced minus oxidized) = 17,500m−1cm−1.

      Stopped Flow

      Compounds P, F, and O were rapidly mixed with saturated (∼2 mm) or diluted solutions of NO, prepared by diluting the stock saturated solution with different volumes of anaerobic buffer, in a stopped flow apparatus (model SX-18MV Applied Photophysics, Leatherhead, UK). The spectra were collected using a photodiode array detector with a time resolution of 3.3 ms. A global fitting analysis program (Global Analysis, Applied Photophysics) using singular value decomposition of the data was used to model the reactions, the spectra of the kinetic components, and the corresponding rate constants. The spectra of the components constructed in this way were consistent with difference spectra obtained by subtracting spectra taken at different time points during the course of the reaction. The amount of CuA reduced after mixing O with NO was obtained by comparing the amplitude of the change at 830 nm with that obtained after the addition of dithionite (
      • Cooper C.E.
      • Torres J.
      • Sharpe M.A.
      • Wilson M.T.
      ).

      Nitric Oxide

      Nitric oxide (NO) was obtained with a Kipps apparatus by mixing 1 m sulfuric acid with sodium nitrite, essentially as described by Torres and Wilson (
      • Torres J.
      • Wilson M.T.
      ). The NO in the stock solution (usually ∼2 mm) and in the dilutions was measured with an NO electrode (Iso-NO Mark II, World Precision Instruments). The level of nitrite in the NO stock solution was also monitored (usually less than 0.1 mm) in the same electrode, by measuring the NO formed after acidification with H2SO4 in the presence of KI.

      RESULTS

      Static Optical Spectroscopy

      Reaction of NO with Species O

      Addition of NO to O generated a difference spectrum (Fig. 1 A) that, in the Soret region, exhibited a peak at 445 nm and a trough at 430 nm. At longer wavelengths, a positive band at 605 nm was also observed. Both the positions of the bands and the relative intensities are indicative of reduction of cytochrome a (∼40% of the total). These changes were accompanied by a bleached region from 620 to 660 nm.
      Figure thumbnail gr1a
      Figure 1Spectral changes occurring on mixing species O, F, and P with 20 μm NO. All difference spectra were recorded with respect to O. A, addition of NO to O;B, a sample containing 80% F before (—–) and after (- - - - -) the addition of NO; C, spectra after the addition of 10 mm sodium nitrite to O (—–) and after the addition of NO to species F (- - - - -) (the visible region has been expanded (×3)); D, a sample containing ∼80% P before (—–) and after (- - - - -) the addition of NO;E, comparison between the spectra (visible region) after addition of NO to the samples O (—–), P (- - - - -), and F (· · · · ·). Concentration of CcO, 4.7 μm. Other details are given under “Materials and Methods.”
      Figure thumbnail gr1b
      Figure 1Spectral changes occurring on mixing species O, F, and P with 20 μm NO. All difference spectra were recorded with respect to O. A, addition of NO to O;B, a sample containing 80% F before (—–) and after (- - - - -) the addition of NO; C, spectra after the addition of 10 mm sodium nitrite to O (—–) and after the addition of NO to species F (- - - - -) (the visible region has been expanded (×3)); D, a sample containing ∼80% P before (—–) and after (- - - - -) the addition of NO;E, comparison between the spectra (visible region) after addition of NO to the samples O (—–), P (- - - - -), and F (· · · · ·). Concentration of CcO, 4.7 μm. Other details are given under “Materials and Methods.”

      Reactions of NO with Species F and P

      Addition of NO to P or F resulted in the rapid depletion of these intermediates. The spectrum resulting from the addition of NO to F (Fig. 1 B) has a peak at 415 nm and a trough at 430 nm in the Soret region, and the region from ∼590 to 660 is bleached. Because the scheme presented above (see Scheme FS1, dotted arrows) predicts the formation of nitrite in the binuclear center of CcO, we tested this prediction by adding nitrite to O and comparing the final spectrum to that obtained after addition of NO to F. Fig. 1 C shows that exposure of O to a high concentration of nitrite (10 mm) generated the same spectral changes as obtained when NO (20 μm) was added to F. The affinity of the enzyme for nitrite is low and required a relatively high nitrite concentration to induce spectral changes of measurable amplitude. The fact that the same effect was obtained with a much lower NO concentration suggests that when NO is added to F, nitrite is formed within the binuclear center and interacts with the oxidized binuclear center (Ob) formed as a result of the conversion F → Ob.
      When NO was added to P (Fig. 1 D), the depletion of this intermediate was accompanied, as for O, by the appearance of bands at 605 and 442 nm, indicative of cytochrome a reduction. However, the extent of the reduction was not, as with O, 40% of the total enzyme, but rather 40% of the P present before the addition of NO (e.g. for a sample containing 50% P, the extent of cytochrome a reduction would be ∼20%).
      The features observed when adding NO to F (Fig. 1 C), assigned to the interaction of nitrite with the oxidized binuclear center, Ob, were also present after addition of NO to the samples O and P (see Fig. 1 E). Indeed, the spectrum resulting after addition of NO to O or P can be shown to be a superposition of two components: (a) interaction of nitrite with Ob, and (b) the reduction of cytochromea (i.e. transition Fea3+→ Fea2+). Accordingly, the subtraction of the spectral contribution of nitrite gives a residual identical to the difference spectrum corresponding to reduced minus oxidized cytochrome a (
      • Liao G.L.
      • Palmer G.
      ) (data not shown). We postulate that NO converts P and F to Ob, and NO reacts with Ob, reducing cytochrome a (Fig. 1 A). However, it is clear that we did not observe cytochrome a reduction after mixing F with NO (Fig. 1 B). This was due to the presence of H2O2 in the sample, because when we repeated the experiment adding catalase prior to mixing F with NO, we observed reduction of cytochrome a simultaneous with the decay of F (not shown). This indicates that H2O2 either impedes the interaction of NO with the binuclear center or reoxidizes cytochrome a as soon as it becomes reduced. It is also worth mentioning that even though CO and H2O2 are still present in solution, neither P nor F were reformed after the reaction of NO with these intermediates. This is consistent with the observed sluggish reactivity of the binuclear center of CcO with H2O2 and with CO (which in certain conditions lead to formation of F and P, respectively; see “Materials and Methods”) after NO has been added to O (not shown).
      Thus, these experiments confirm three predictions derived from SchemesFS1 and FS2. First, the product of the reaction of NO with O, P, or F displays a spectrum consistent with the presence of nitrite interacting with the oxidized binuclear center (Ob). Second, the interaction of Ob with NO ejects an electron to cytochromea. Third, P and F are readily depleted by NO. These reactions were fast and no time courses could be observed. We therefore performed similar experiments in a stopped flow apparatus equipped with a diode array detector.

      Stopped Flow Spectrophotometry

      Reaction of Compound F with NO

      The reaction of a sample containing compound F (∼85%) with NO led, as expected (see Fig. 1 B), to the rapid decay of this compound (Fig. 2, curves a and b). The time course was fitted to a biexponential decay, and the rates of the two phases were essentially proportional to the NO concentration (see Fig. 2 legend). Difference spectra relative to O are shown in Fig. 2, inset. The final spectrum was found to be identical to that obtained in the static spectral experiments (Fig. 1 B) and thus identical to that observed after the addition of nitrite to the oxidized enzyme (Fig. 1 C). Although the time course could be fitted to a biexponential decay, the spectra corresponding to the two kinetic components were indistinguishable (not shown). This indicates that the components corresponding to the decay of compound F and the appearance of the features that we identify with the interaction of the binuclear center with nitrite cannot be separated in this reaction.
      Figure thumbnail gr2
      Figure 2Time course and spectral changes observed after mixing cytochrome c oxidase containing F with NO. Species F was mixed with a 1 mm (a) or 400 μm (b) solution of NO. The time courses were fitted to the sum of two exponentials curve: a,k1 = 27.1 s−1 and k2 = 5 s−1; b,k1 = 11 s−1 and k2 = 2.5 s−1. Inset, difference spectra versus O. Δt = 10 ms (spectra 1–10) and 100 ms (spectra 11–20). The arrowsindicate the direction of the changes. λ = 422 nm is isosbestic for the transition. CcO concentration, 4 μm after mixing.

      Reactions of Compound P with NO

      Reactions within the Dead Time (1.4 ms)

      When a sample containing a high proportion of compound P (∼80%) was mixed with NO in the stopped flow spectrophotometer, some spectral changes took place in the dead time of the apparatus (∼1.4 ms), whereas the band at 607 nm remained unchanged in this time frame (Fig. 3, arrows). The final spectrum after ∼1 s is consistent with that shown in Fig. 1 D(static experiments), showing reduction of cytochrome a and depletion of P. The difference spectrum corresponding to the changes which occurred during the dead time is given in Fig. 4 A, solid line (and also in Fig. 3, inset, solid line). This spectrum (Fig. 4 A, solid line) shows two peaks in the Soret region at about 425 and 445 nm and a trough around 410 nm. At longer wavelengths, a positive band at ∼580 nm, a shoulder at 600 nm, and a negative region centered at ∼615 nm can also be observed. These features are reminiscent of the formation of compound F, possibly accompanied by some reduction of cytochrome a. However, both the changes in the Soret and the α regions are slightly different from those expected for simultaneous formation of F and reduction of cytochromea. For example, although a band at 445 nm is clearly present in the Soret region, there is no concomitant increase in the α band at ∼605 nm. Only a shoulder at 600 nm is observed. We can discount spectral contributions from a transition of the type P → F, because the increase at 580 nm is not paralleled by a decrease at 607 nm of an amplitude approximately twice as large (see the extinction coefficients given under “Materials and Methods”) (Fig. 4 A, solid line). In addition, even in case that reduction of cytochromea (increase at 605 nm) compensates for the decrease at 607 nm, the change at 445 nm in the Soret region (which is 3 times larger in amplitude than at 605 nm; see Fig. 1 A) would have to be about twice as large than observed in Fig. 4 A.
      Figure thumbnail gr3
      Figure 3Spectral changes observed upon mixing P with NO. Difference spectra relative to O generated on mixing P with 1 mm NO. —–, before mixing; · · · · ·, 1.4 ms after mixing; - - - - -, 800 ms after mixing. Inset, difference spectra corresponding to the changes occurring in the dead time (i.e. 1.4 ms after mixing minus before mixing) (—–) and the changes occurring between 1.4 ms and 800 ms after mixing (- - - - -). CcO concentration, 2.35 μm after mixing; P was 80%.
      Figure thumbnail gr4
      Figure 4Reactions of samples containing P with NO: changes occurring in the spectrophotometer dead time (1.4 ms). A, spectral changes occurring on mixing P (2.35 μm CcO after mixing) with NO (—–) and those observed after the addition of 10 mm nitrite to O (- - - - -). B, difference spectra obtained after the contribution of nitrite binding (—–) and the contributions of both nitrite binding and reduction of cytochrome a (positive bands at 605 nm and 445 nm) (- - - - -) were subtracted from the difference spectrum depicted as a solid line inA. The difference spectrum for the reduction of cytochromea was obtained as described under “Materials and Methods.”
      Thus, a transition of the type O → F seems more plausible, except that the peak in the Soret region, which should appear at ∼435 nm (
      • Fabian M.
      • Palmer G.
      ), appears to be shifted to 425 nm. These differences indicate that other changes, possibly attributable to the effect of nitrite (see Fig. 1 C), distort the composite spectrum arising from the formation of compound F and the reduction of cytochrome a. In fact, when the contribution of the interaction of nitrite with Ob (Fig. 4 A, dotted line) was subtracted, the spectral features conformed more closely to those expected from the transitions O → F and Fea3+ → Fea2+ (Fig. 4 B). Further, when the contribution of the reduction of cytochrome a was also subtracted (see Fig. 4 legend for details), the resulting spectrum was very similar to that of F relative to O (see Fig. 1 B, solid line). This analysis reveals that when NO is mixed with samples containing P, spectral changes occur within the dead time of the stopped flow spectrometer consistent with the following events: (a) formation of species F from a species spectrally similar to O, not from P, (b) formation of nitrite within the binuclear center, and (c) reduction of some cytochromea.
      When P was generated from mixed valence-CO enzyme (∼80% P), the percentage of compound F formed in the dead time was only ∼20% of the total enzyme. However, when P was generated by bubbling CO in the presence of oxygen (∼40% P), the percentage of F generated was larger (40–50%) (
      • Torres J.
      • Wilson M.T.
      ). This indicates that the precursor of F is not P itself. This finds further support from experiments in which compound P, generated from the mixed valence-CO preparation, was allowed to decay at room temperature (not shown) prior to mixing with NO. Addition of NO after a long period (∼1 h) produced a larger fraction of F formed (30–40%) in the dead time. Only after about 2 h was a fast partial reduction of cytochrome a (k∼ 100 s−1), which corresponded to ∼20% of the enzyme (not shown), also observed, consistent with a proportion of the molecules having returned to O. This shows that the species absorbing at 607 nm does not decay directly to O but rather to another species that is O-like in spectral properties but that generates F rapidly on mixing with NO.

      Reactions Following the Dead Time

      Following the dead time, species P (607 nm) and the newly formed species absorbing at 580 nm decayed simultaneously (Fig. 5), with a rate that was found to depend on the NO concentration (Fig. 5,inset). These changes were complete in ∼800 ms at 1 mm NO, and together with those changes observed in the Soret region, they are compatible with the decay of compounds P and F. The amplitude of the decrease at 580 nm from 1.4 to 800 ms was found to be identical, within our experimental error, to the amplitude of the increase observed within the dead time (Fig. 3, inset). This indicates that the compound F that decays from 1.4 to 800 ms corresponds to that formed within the dead time. The amplitude of the decrease at 607 nm, however, was smaller than the corresponding amplitude of the P present before mixing (Fig. 3). In fact, one of the troughs in Fig. 5 is centered at 612 nm, not at 607 nm. This apparent shift can be explained by a simultaneous decrease at 607 nm and an increase at 605 nm, which, together with the presence of a positive band at 442 nm in the difference spectrum, indicates that some reduction of cytochrome a takes place simultaneously with the decay of the P and F intermediates.
      Because the spectra of P and reduced cytochromea are similar in the α band (P has a band at 607 nm and cytochrome a at 605 nm) the simultaneous decay of P and a smaller increase of reduced cytochrome a led only to a small perturbation in this region (see extinction coefficients under “Materials and Methods”).
      Accordingly, the reduction of cytochrome a and the decay of P and F resulted in a single kinetic transition in the global analysis (see “Materials and Methods”). In addition, internal differences between spectra collected at different time points show that the component corresponding to cytochrome a reduction is present at all time intervals (not shown). This indicates that both processes are interconnected, such that the rate of reduction of cytochromea is limited by the rate of decay of the intermediates P or F. As in the static experiments (Fig. 1 D), the final amount of cytochrome a reduced was about 40% of the P initially present.
      Figure thumbnail gr5
      Figure 5Spectral changes obtained after mixing CcO samples containing P with NO. CcO (2.35 μm after mixing) containing ∼80% P was mixed with NO (1 mm after mixing). Difference spectra relative to the first spectrum collected after mixing (1.4 ms) are shown at a number of times during the time course of the reaction. Time points: first spectrum, 4ms; spectra 2–10, Δt = 10 ms; spectra 10–18, Δt = 100 ms. The arrows indicate the direction of the changes; the region 500–800 has been expanded (×5), and characteristic wavelengths are indicated. Inset, time courses of the absorbance changes monitored as ΔA(430–500 nm) after mixing P (as above) with 1 mm (a) or 20 μm (b) NO. The time courses (—–) were fitted to a monoexponential decay (○), and the rate constants for the decay are indicated.

      Reactions of O with NO

      As for compound P, when NO was mixed with O some changes occurred within the instrumental dead time (<1.4 ms) (Fig. 6 A). The difference spectrum recorded (t = 1.4 ms minus t = 0 ms) is compatible with the appearance of approximately equal percentage (∼15%) of reduced cytochrome a and compound F. Although the reduction of cytochrome a observed may be expected (in ∼1.4 ms, a process with k = 100 s−1completes ∼15% of its total amplitude), the formation of F was not anticipated. We attribute the formation of F to heterogeneity in the sample, suggesting that preparations of O (after reduction and reoxidation; see “Materials and Methods”) contain a small proportion of the O-like species
      Other authors have previously suggested the presence of heterogeneities even in “fast” oxidase preparations, based on the presence of two phases in the kinetics of ligand binding (
      • Moody A.J.
      • Cooper C.E.
      • Rich P.R.
      ), especially formate.
      (described above). After the dead time, about 40% of cytochrome a became reduced (k ∼ 100 s−1), as indicated by bands at 442 and 605 nm (intensity ratio, 3:1) (Fig. 6 B), in agreement with the results obtained in the static spectral experiments (Fig. 1 A). Reduction of cytochrome a was accompanied by reduction of 15–20% CuA (not shown) (
      • Cooper C.E.
      • Torres J.
      • Sharpe M.A.
      • Wilson M.T.
      ).
      Figure thumbnail gr6
      Figure 6Spectral changes after mixing O with NO. A, difference spectra between that obtained 1.4 ms after mixing and the original spectrum of O (—–) and after the contribution of reduction of cytochrome a was subtracted (- - - - -). This spectrum is consistent with the formation of compound F.B, difference spectra relative to the spectrum collected 3.8 ms after mixing (Δt = 4 ms). C, difference spectra relative to the spectrum collected 50 ms after mixing (t = 55, 70, 130, 400, 580, 750, and 950 ms after mixing). Conditions: O, 5 μm; NO, 1 mm after mixing.
      In a slower phase (Fig. 6 C), a decay at 580 nm and a simultaneous increase at 605 nm was also observed (k∼ 8 s−1). Both these changes and those detected in the Soret region are indicative of simultaneous decay of F (formed in the dead time) and reduction of cytochrome a. This slow process is similar to that seen when mixing P and NO, when reduction of cytochrome a accompanied the decay of the bands corresponding to P (607 nm) (Fig. 5). This can be clearly seen in Fig. 7, where the difference spectrum produced ∼50 ms after mixing O with NO (i.e. at the end of the process with k ∼ 100 s−1) (see Fig. 6 B), is compared with the difference spectrum of the final product (after ∼800 ms) obtained when mixing P with NO (i.e. following the process with k ∼ 8 s−1) (from Fig. 3). It is clear from this figure that the same optical change is obtained in both cases, regardless of the initial species (P or O), although with different kinetics. Together with the results in Fig. 6 C, these results indicate that the reduction of cytochrome a is rate limited by the decay of P or F when these oxygen intermediates are present.
      Figure thumbnail gr7
      Figure 7Comparison between the difference spectra obtained on mixing NO with P or O. Difference spectra with respect to the oxidized enzyme O obtained on mixing with 1 mm NO either O (- - - - -) or a sample containing ∼80% P (—–). The spectra were collected 55 ms and 1 s after mixing NO with O and P, respectively. CcO, 4 μm after mixing.

      DISCUSSION

      The results presented above may be rationalized by reference to a simplified reaction mechanism presented in SchemeFS3. The basic feature of this scheme is that the different reactions, observed upon mixing NO with derivatives of CcO, can all be explained by a single electron donation from NO to the binuclear center. Each step in Scheme FS3 comprises a sequence of events analogous to those indicated by dotted arrows in Scheme FS1, i.e. NO binds to CuB2+ and results in the formation of HNO2 and reduction of CuB. The latter would permit electron transfer from CuB either to cytochromea/CuA or to oxygen intermediates bound to Fea3. A detailed reaction mechanism is given in SchemeFS4, in which the two parts, A and B, differ in the chemical assignment of P. We have assumed in part A that the species P (607 nm) and F (580 nm) are the ferric peroxy and oxoferryl derivatives of Fea3, respectively, as suggested previously by Wikström and Morgan (
      • Wikström M.
      • Morgan J.E.
      ).
      Because we propose that NO provides the binuclear center with one reducing equivalent, it follows that the precursor of the F formed during the dead time (spectrally O-like and labeled X in Scheme FS4) is at the formal oxidation level of peroxide. Therefore, if P (PM) is the ferric peroxy species, then X could contain either CuB3+, with the iron as a ferryl oxo species (Fe4+ = O), or alternatively as CuB2+, with Fe5+ = O (see SchemeFS4). The presence of either of these species, suggested previously by other authors (
      • Fabian M.
      • Palmer G.
      ,
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S.
      • Appelman E.H.
      • Kitagawa T.
      ,
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S
      • Kitagawa T.
      ,
      • Verkhovsky M.I.
      • Morgan J.E.
      • Wikström M.
      ,
      • Ogura T.
      • Hirota S.
      • Proshlyakov D.A.
      • Shinzawa-Itoh K.
      • Yoshikawa S.
      • Kitagawa T.
      ,
      • Babcock G.T.
      • Floris R.
      • Nilsson T.
      • Pressler M.
      • Varotsis C.
      • Vollenbroek E.
      ), would explain the fast formation of compound F solely by reduction of CuB3+ to CuB2+ or Fe5+ to Fe4+. Our data suggest that PM coexists with species X. possibly in an equilibrium, because they are at the same oxidation level.
      An analogous and equally fast reaction may be expected to occur between NO and PM, generating a peroxy species with CuBreduced. Such a species, postulated previously, has been termed PR in the literature (
      • Morgan J.E.
      • Verkhovsky M.I.
      • Wikström M.
      ).
      PR is the form obtained after photolysis, in the presence of oxygen, of the Fea3—CO bond in CO-bound fully reduced oxidase. In this case, the second reducing equivalent for oxygen is provided directly from cytochromea, and not by CuB, which remains reduced. In contrast, the species obtained from CO-bound mixed valence enzyme, in which the second reducing equivalent for oxygen is provided through oxidation of CuB, is called PM (
      • Morgan J.E.
      • Verkhovsky M.I.
      • Wikström M.
      ).
      The conversion from PM to PR would not result in observable optical changes, because these species have similar spectra (
      • Morgan J.E.
      • Verkhovsky M.I.
      • Wikström M.
      ). This is in agreement with our results, because the band at 607 nm corresponding to P remains unchanged on addition of NO during the dead time (Fig. 3). Thus, after mixing a sample containing P (PM and X) with NO, we obtained two different products: one was PR, from the transition PM → PR, and the other was F, from the transition X → F. The mechanism in Scheme FS4 accounts, therefore, for the spectral changes that occur within the dead time after mixing P with NO, namely, the apparent nonreactivity of PM (Fig. 5) and the formation of F from a species spectrally similar to O.
      Furthermore, our results show that the species F (absorbing at 580 nm) and PR (assumed to absorb at 607 nm) (
      • Morgan J.E.
      • Verkhovsky M.I.
      • Wikström M.
      ) decay simultaneously after mixing with NO (Fig. 5). This suggests that they are in rapid equilibrium, one of them reacting with NO and recruiting the other into the reactive form. From our experiments (Fig. 1 B), it is clear that it is compound F that reacts with NO yielding O, and we thus incorporated this into Scheme FS4. Thus, PR would react not with NO per se, but through an equilibrium established between PR and F. For the reaction between F and NO, a reaction analogous to that described for PM and species X was suggested, after which, an internal electron transfer from CuB could facilitate the transition F → Ob. Support for the role of CuB in this reaction comes from the fact that although F is known to react with CO (
      • Blair D.F.
      • Witt S.N.
      • Chan S.I.
      ) and other ferryl groups (e.g. in myoglobin) react with NO (

      Gorbunov, N. V., Osipov, A. N., Day, B. W., Zayas-Rivera, B., Kagan, V. E., and Elsayed, N. M. (1995)34, 6689–6699.

      ), these processes are much slower than those presented here. After the formation of species O, a further electron donation from NO would then lead to the observed reduction of cytochrome a. In agreement with this model, our results show that this process is fast when mixing O with NO (k = 100 s−1) but is rate-limited by the decay of P and F (∼8 s−1) when NO reacts with these intermediates. This is expected if the substrate for this reduction (Ob) is the product of the reaction of P and F with NO, as Scheme FS4 shows.

      The Identity of Compound X

      It seems generally agreed that F (580 nm) has an oxo ferryl structure, containing CuB2+, but the assignment of P (607 nm) is more problematic. Basically, the discussion in the literature revolves around whether the oxygen O—O bond can be broken following addition of only two electrons to the enzyme. This could be achieved by means of an additional electron, recruited from elsewhere in the protein, to produce an oxo ferryl derivative. Four main sources for this electron have been suggested, i.e. the porphyrin ring, an amino acid, the copper atom CuB, or the iron atom itself. The donation of an electron from the porphyrin ring or an amino acid, leading to the formation of a radical, as observed in other enzymes (i.e.peroxidases and catalases), seems to be unlikely (
      • Fabian M.
      • Palmer G.
      ,
      • Ogura T.
      • Hirota S.
      • Proshlyakov D.A.
      • Shinzawa-Itoh K.
      • Yoshikawa S.
      • Kitagawa T.
      ,
      • Morgan J.E.
      • Verkhovsky M.I.
      • Wikström M.
      ), although a radical has been observed to occur in the enzyme in turnover (
      • Wilson M.T.
      • Jensen P.
      • Aasa R.
      • Malström B.G.
      • Vangaard T.
      ). The assignment of the 607 nm form to an oxoferryl Fe4+= O structure with CuB3+ has been postulated by other authors (
      • Ferguson-Miller S.
      • Babcock G.T.
      ,
      • Fabian M.
      • Palmer G.
      ,
      • Babcock G.T.
      • Floris R.
      • Nilsson T.
      • Pressler M.
      • Varotsis C.
      • Vollenbroek E.
      ), and this unusually high oxidation state for CuB has been recently detected in an inorganic model (
      • Cole A.P.
      • Root D.E.
      • Mukherjee P.
      • Solomon E.I.
      • Stack T.D.P.
      ). Proshlyakov et al. (
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S.
      • Appelman E.H.
      • Kitagawa T.
      ) also associate the 607 nm absorbing species to an iron oxo group on the basis of Resonance Raman16O-18O mixed-isotope experiments with 607 nm excitation. These authors, however, attribute the band that appears at 804 cm−1 to Fe5+ = O (and CuB2+).
      We propose that X could be represented by either of these two species. In our model, species X has a spectrum more similar to O than to P. This is in contrast with the conclusions of Morgan et al.(
      • Morgan J.E.
      • Verkhovsky M.I.
      • Wikström M.
      ), who argued against the formation of CuB3+at the peroxide level. However, their conclusions were based on the assumption that the spectrum of the compound Fe4+ = O CuB3+ would be identical to that of P (607 nm). In addition, the parallel increase of the Raman band at 804 cm−1 and the optical band at 607 nm (
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S
      • Kitagawa T.
      ), could correspond to two different species, the parallel increase of these two signals being due to an equilibrium between X (optically O-like) and PM (607 nm) (see Scheme FS4, top equilibrium).
      The postulated interconversion of the species PR and F may also be considered in the context of the Raman data, e.g.the assignment of the band at 356 cm−1. Proshlyakovet al. (
      • Proshlyakov D.A.
      • Ogura T.
      • Shinzawa-Itoh K.
      • Yoshikawa S.
      • Kitagawa T.
      ) assigned this band to a bending mode of the unusual Fe5+ = O species (referred to above) on the basis that the oxo oxygen could be exchanged with the oxygen in water through a reversible formation of ferric hydroxy (Fe3+OH). An alternative view is that the species absorbing at 356 cm−1 is analogous to either PR (
      • Varotsis C.
      • Zhang Y.
      • Appelman E.H.
      • Babcock G.T.
      ,
      • Varotsis C.
      • Babcock G.T.
      ) or a ferrous peroxy/cupric species (Fe2+O22− CuB2+) (
      • Ferguson-Miller S.
      • Babcock G.T.
      ), as depicted in Scheme FS4 (part A). These species are in equilibrium with F, explaining the oxygen exchange data. Interestingly, a fast equilibrium between species PR and F, as depicted in Scheme FS4, has recently been suggested to exist during the transition, initiated by photolysis at room temperature, from fully reduced CO-bound CcO to the oxidized enzyme (
      • Sucheta A.
      • Georgiadis K.E.
      • Einarsdóttir Ó.
      ). Obviously, this equilibrium implies that the splitting of the O—O bond is reversible in these conditions, and this poses a problem from the thermodynamic point of view.
      This problem can be avoided as shown in Scheme FS4 (part B), where PM and PR are assigned the structures Fe5+ = O, with CuB2+ and CuB1+, respectively, instead of ferric peroxide species. In this case, PR and F are also in the same oxidation state, but there is no need for reversible O—O cleavage. Clearly, this model lacks the ferric peroxy form, in contradiction of previous assignments and observations made using synthetic model compounds in which a peroxy species was detected during the the reduction of oxygen to water (
      • Collman J.P.
      • Fu L.
      • Herrmann P.C.
      • Zhang X.
      ).
      The results presented indicate that a species (X) spectrally similar to O is in equilibrium with the 607 nm species in the sample containing compound P. In our view, this could clarify some of the apparent inconsistencies concerning the structural assignments of the intermediates P and F. For example, the cytochrome iron structure in compound P has been described either as a ferric peroxy or an iron oxo (Fe4+ = O or Fe5+ = O) by different authors (see references under “Introduction”). This discrepancy could be explained if a ferric peroxy and an iron oxo (either Fe4+ = O or Fe5+ = O, as shown in Scheme FS4, part A), or two iron oxo species (e.g. Fe4+ = O and Fe5+ = O, as in Scheme FS4, part B) coexisted at the peroxide level. Thus, in Scheme FS4, part A, the ferric peroxy (PM) would be the species detected optically (at 607 nm), whereas either of the postulated as species X (Fe4+ = O with CuB3+ or Fe5+ = O with CuB2+) could be detected at 804 cm−1 by Raman. Alternatively, in Scheme FS4, part B, either species X (Fe4+ = O with CuB3+) or PM (Fe5+ = O with CuB2+) could be detected by the iron oxo stretching νFe= O vibration at 804 cm−1with Raman spectroscopy, but it is possible that Fe5+ = O may also be detected optically by difference spectroscopy (at 607 nm). In addition, it is worth noting that the assignment of species X as Fe4+ = O with CuB3+ is compatible with both parts A and B in Scheme FS4, and as such, it may appear to be the more likely candidate. On the other hand, as discussed above, the experimental results suggesting that PR and F are in rapid equilibrium are better explained if PR and PM are both iron oxo (Fe5+ = O), supporting the assignments for X and PM made inpart B of Scheme FS4.
      A further question concerns the fate of the electrons ejected from the binuclear center. In our experiments with the oxidized enzyme, O, approximately 50% of cytochrome a and ∼15–20% of CuA became reduced (
      • Cooper C.E.
      • Torres J.
      • Sharpe M.A.
      • Wilson M.T.
      ). Furthermore, approximately 20% of F was formed within the dead time. This suggests that around 90% of the molecules became reduced with one electron, and this is in agreement with recent experiments that show that only three electrons could be donated from cytochrome c to CcO prior to its inhibition by NO (
      • Giuffre A.
      • Sarti P.
      • D'Itri E.
      • Buse G.
      • Soulimane T.
      • Brunori M.
      ).

      Physiological Implications of the Reactions Observed between NO and CcO

      Our finding that nitrite is rapidly formed in the binuclear center of CcO suggests that Scheme FS1 is likely to be one of the pathways through which NO is metabolized and detoxified. In addition, this mechanism is consistent with the fact that mitochondrial NO metabolism by CcO also occurs anaerobically (
      • Borutaité V.
      • Brown G.C.
      ), because the mechanism in Scheme FS1 does not require oxygen. These results also have implications regarding the mechanism of inhibition of CcO by NO. It has been suggested that this process can be explained by reversible binding of NO to ferrous cytochromea3 (
      • Giuffre A.
      • Sarti P.
      • D'Itri E.
      • Buse G.
      • Soulimane T.
      • Brunori M.
      ), and this is consistent with the competitive nature of the inhibition with respect to oxygen. However, consideration of the “on” and “off” rates of NO with ferrous a3 (
      • Blackmore R.S.
      • Greenwood C.
      • Gibson Q.H.
      ,
      • Giuffre A.
      • Sarti P.
      • D'Itri E.
      • Buse G.
      • Soulimane T.
      • Brunori M.
      ) is not in agreement with the observed lowKi for the inhibition of cellular respiration by NO (
      • Borutaité V.
      • Brown G.C.
      ). This apparent conflict can be explained if, as we have shown, an additional interaction of NO with the binuclear center (i.e.with CuB2+) takes place, thus decreasing theKi of NO for CcO. Thus, although formation of ferrous a3-nitrosyl complex has been detected in the inhibitory process (
      • Torres J.
      • Darley-Usmar V.M.
      • Wilson M.T.
      ) and has been suggested as the primary event in the inhibition (
      • Giuffre A.
      • Sarti P.
      • D'Itri E.
      • Buse G.
      • Soulimane T.
      • Brunori M.
      ), this work suggests that formation of ferrocytochrome a3 may be a consequence rather than a cause of the inhibition. A possible mechanism by which inhibition could take place is suggested by the fast reduction of CuB2+ to CuB1+ by NO. The competition of NO and oxygen would not be at the ferrocytochrome a3 but instead at CuB1+. Furthermore, binding of NO to CuB1+ could block the redox state of this metal, which would then constitute the real inhibitory site. Alternatively, NO may transfer rapidly from CuB1+ to cytochrome a3 on addition of an electron to this site in turnover.

      Acknowledgment

      We acknowledge M. Sharpe for helpful discussions.

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