Resolution of electrogenic steps coupled to conversion of cytochrome c oxidase from the peroxy to the ferryl-oxo state

Charge translocation across the membrane coupled to transfer of the third electron in the reaction cycle of bovine cytochrome c oxidase (COX) has been studied. Flash-induced reduction of the peroxy intermediate (P) to the ferryl-oxo state (F) by tris-bipyridyl complex of Ru(II) in liposome-reconstituted COX is coupled to several phases of membrane potential generation that have been time-resolved with the use of an electrometric technique applied earlier in the studies of the ferryl-oxo-to-oxidized (F --> O) transition of the enzyme [Zaslavsky, D., et al. (1993) FEBS Lett. 336, 389-393]. As in the case of the F --> O transition, the electric response associated with photoreduction of P to F includes a rapid KCN-insensitive electrogenic phase with a tau of 40-50 microseconds (reduction of heme a by CuA) and a multiphasic slower part; this part is cyanide-sensitive and is assigned to vectorial transfer of protons coupled to reduction of oxygen intermediate in the binuclear center. The net KCN-sensitive phase of the response is approximately 4-fold more electrogenic than the rapid phase, which is similar to the characteristics of the F --> O electrogenic transition and is consistent with net transmembrane translocation of two protons per electron, including vectorial movement of both "chemical" and "pumped" protons. The protonic part of the P --> F electric response is faster than in the F --> O transition and can be deconvoluted into three exponential phases with tau values varying for different samples in the range of 0.25-0.33, 1-1.5, and 6-7.5 ms at pH 8. Of these three phases, the 1-1.5 ms component is the major one contributing 50-60%. The P --> F conversion induced by single electron photoreduction of the peroxy state as studied in this work is several times slower than the P --> F transition resolved during oxidation of the fully reduced oxidase by molecular oxygen. The role of the CuB redox state in controlling the rate of P --> F conversion of heme a3 is discussed.     

Electrogenic reactions of cytochrome bd

Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O(2) and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (delta psi, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 mus that matches the formation of ferryl-oxo heme d, the product of the reaction of O(2) with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H(2)O(2) with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a delta psi of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b(558) and backward. The amplitude of the delta psi produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O(2) and is apparently associated with full transmembrane translocation of approximately one charge.     

An elementary reaction step of the proton pump is revealed by mutation of tryptophan-164 to phenylalanine in cytochrome c oxidase from Paracoccus denitrificans

Cytochrome c oxidase couples reduction of dioxygen to water to translocation of protons over the inner mitochondrial or bacterial membrane. A likely proton acceptor for pumped protons is the Delta-propionate of heme a(3), which may receive the proton via water molecules from a conserved glutamic acid (E278 in subunit I of the Paracoccus denitrificans enzyme) and which receives a hydrogen bond from a conserved tryptophan, W164. Here, W164 was mutated to phenylalanine (W164F) to further explore the role of the heme a(3) Delta-propionate in proton translocation. FTIR spectroscopy showed changes in vibrations possibly attributable to heme propionates, and the midpoint redox potential of heme a(3) decreased by approximately 50 mV. The reaction of the oxidized W164F enzyme with hydrogen peroxide yielded substantial amounts of the intermediate F' even at high pH, which suggests that the mutation rearranges the local electric field in the binuclear center that controls the peroxide reaction. The steady-state proton translocation stoichiometry of the W164F enzyme dropped to approximately 0.5 H(+)/e(-) in cells and reconstituted proteoliposomes. Time-resolved electrometric measurements showed that when the fully reduced W164F enzyme reacted with O(2), the membrane potential generated in the fast phase of this reaction was far too small to account either for full proton pumping or uptake of a substrate proton from the inside of the proteoliposomes. Time-resolved optical spectroscopy showed that this fast electrometric phase occurred with kinetics corresponding to the transition from state A to P(R), whereas the subsequent transition to the F state was strongly delayed. This is due to a delay of reprotonation of E278 via the D-pathway, which was confirmed by observation of a slowed rate of Cu(A) oxidation and which explains the small amplitude of the fast charge transfer phase. Surprisingly, the W164F mutation thus mimics a weak block of the D-pathway, which is interpreted as an effect on the side chain isomerization of E278. The fast charge translocation may be due to transfer of a proton from E278 to a "pump site" above the heme groups and is likely to occur also in wild-type enzyme, though not distinguished earlier due to the high-amplitude membrane potential formation during the P(R)--> F transition.     

Mutation of Arg-54 strongly influences heme composition and rate and directionality of electron transfer in Paracoccus denitrificans cytochrome c oxidase

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.     

Single-electron photoreduction of the P(M) intermediate of cytochrome c oxidase

The P(M)-->F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P(M) state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is approximately 5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F-->O transition, but is significantly slower than formation of the F state from the P(R) intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70-90 micros). The approximately 0.3 ms P(M)-->F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P(M)-->F transition is generated much later, with a time constant of 1.3 ms. In addition, the P(M)-->F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with tau values of 0.18 and 0.85 ms.     

Transmembrane charge separation during the ferryl-oxo -> oxidized transition in a nonpumping mutant of cytochrome c oxidase

The N139D mutant of cytochrome c oxidase from Rhodobacter sphaeroides retains full steady state oxidase activity but completely lacks proton translocation coupled to turnover in reconstituted liposomes (Pawate, A. S., Morgan, J., Namslauer, A., Mills, D., Brzezinski, P., Ferguson-Miller, S., and Gennis, R. B. (2002) Biochemistry 41, 13417-13423). Here, time-resolved electron transfer and vectorial charge translocation in the ferryl-oxo --> oxidized transition (transfer of the 4th electron in the catalytic cycle) have been studied with the N139D mutant using ruthenium(II)-tris-bipyridyl complex as a photoactive single-electron donor. With the wild type oxidase, the flash-induced generation of Deltaphi in the ferryl-oxo --> oxidized transition begins with rapid vectorial electron transfer from CuA to heme a (tau approximately 15 micros), followed by two protonic phases, referred to as the intermediate (0.4 ms) and slow electrogenic phases (1.5 ms). In the N139D mutant, only a single protonic phase (tau approximately 0.6 ms) is observed, which was associated with electron transfer from heme a to the heme a3/CuB site and decelerates approximately 4-fold in D2O. With the wild type oxidase, such a high H2O/D2O solvent isotope effect is characteristic of only the slow (1.5 ms) phase. Presumably, the 0.6-ms electrogenic phase in the N139D mutant reports proton transfer from the inner aqueous phase to Glu-286, replacing the "chemical" proton transferred from Glu-286 to the heme a3/CuB site. The transfer occurs through the D-channel, because it is observed also in the N139D/K362M double mutant in which the K-channel is blocked. It is concluded that the intermediate electrogenic phase observed in the wild type enzyme is missing in the N139D mutant and is because of translocation of the "pumped" proton from Glu-286 to the D-ring propionate of heme a3 or to release of this proton to the outer aqueous phase. Significantly, with the wild type oxidase, the protonic electrogenic phase associated with proton pumping (approximately 0.4 ms) precedes the electrogenic phase associated with the oxygen chemistry (approximately 1.5 ms).     

Acceleration of the oxygen reaction in CuA-deficient Nitrosomonas europaea cytochrome c oxidase as revealed by the flow-flash measurement.

The oxygen reaction of Nitrosomonas europaea cytochrome c oxidase containing either 2Cu or 1Cu per two heme a molecules was investigated by the flow-flash technique at 20 degrees C. The reaction profiles of the bacterial enzyme were essentially the same as those of bovine heart cytochrome c oxidase, although the rate of the primary oxygen compound formation was much slower. The 1Cu enzyme exhibited higher rates for both primary oxygen compound formation and intramolecular electron transfer than the 2Cu enzyme. This result clearly indicates that CuA is not essential functionally for the oxidation of ferrous heme a moieties, and suggests its structural importance in maintaining the molecular integrity of N. europaea cytochrome oxidase.     

Elementary steps of proton translocation in the catalytic cycle of cytochrome oxidase

Proton translocation in the catalytic cycle of cytochrome c oxidase (CcO) proceeds sequentially in a four-stroke manner. Every electron donated by cytochrome c drives the enzyme from one of four relatively stable intermediates to another, and each of these transitions is coupled to proton translocation across the membrane, and to uptake of another proton for production of water in the catalytic site. Using cytochrome c oxidase from Paracoccus denitrificans we have studied the kinetics of electron transfer and electric potential generation during several such transitions, two of which are reported here. The extent of electric potential generation during initial electron equilibration between CuA and heme a confirms that this reaction is not kinetically linked to vectorial proton transfer, whereas oxidation of heme a is kinetically coupled to the main proton translocation events during functioning of the proton pump. We find that the rates and amplitudes in multiphase heme a oxidation are different in the OH-->EH and PM-->F steps of the catalytic cycle, and that this is reflected in the kinetics of electric potential generation. We discuss this difference in terms of different driving forces and relate our results, and data from the literature, to proposed mechanisms of proton pumping in cytochrome c oxidase.     

Proton and electron pathways in the bacterial nitric oxide reductase

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.     

Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen.

To probe the functional role of a bound ubiquinone-8 in cytochrome bo-type ubiquinol oxidase from Escherichia coli, we examined reactions with ubiquinol-1 and dioxygen. Stopped-flow studies showed that anaerobic reduction of the wild-type and the bound ubiquinone-free (DeltaUbiA) enzymes with ubiquinol-1 immediately takes place with four kinetic phases. Replacement of the bound ubiquinone with 2,6-dibromo-4-cyanophenol (PC32) suppressed the anaerobic reduction of the hemes with ubiquinol-1 by eliminating the fast phase. Flow-flash studies in the reaction of the fully reduced enzyme with dioxygen showed that the heme b-to-heme o electron transfer occurs with a rate constant of approximately 1x10(4) s(-1) in all three preparations. These results support our previous proposal that the bound ubiquinone is involved in facile oxidation of substrates in subunit II and subsequent intramolecular electron transfer to low-spin heme b in subunit I.     

Membrane tetraheme cytochrome c(m552) of the ammonia-oxidizing nitrosomonas europaea: a ubiquinone reductase

Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.     

Electron transfer rates and equilibrium within cytochrome c oxidase.

Intramolecular electron transfer (ET) between the CuA center and heme a in bovine cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methyl nicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 830 nm. After the initial reduction phase, the 830 nm absorption was partially restored, corresponding to reoxidation of the CuA center. Concomitantly, the absorption at 445 nm and 605 nm increased, indicating reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration. This demonstrates that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse (heme a --> CuA) process were found to be 13 000 s-1 and 3700 s-1, respectively, at 25 degrees C and pH 7.4. This corresponds to an equilibrium constant of 3.4 under these conditions. Thermodynamic and activation parameters of the ET reactions were determined. The significance of these results, particularly the observed low activation barriers, are discussed within the framework of the known three-dimensional structure, ET pathways and reorganization energies.     

Rates and Equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase

Intramolecular electron transfer between CuA and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the CuA center and the concomitant reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse heme a --> CuA process were found to be 20,400 s(-1) and 10,030 s(-1), respectively, at 25 degrees C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme's known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.     

A new ruthenium complex to study single-electron reduction of the pulsed O(H) state of detergent-solubilized cytochrome oxidase

The first step in the catalytic cycle of cytochrome oxidase, the one-electron reduction of the fully oxidized enzyme, was investigated using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine)2]2(quaterpyridine), (Ru2Z). The aim of the work was to examine differences in the redox kinetics resulting from pulsing the oxidase (i.e., fully reducing the enzyme followed by reoxidation) just prior to photoreduction. Recent reports indicate transient changes in the redox behavior of the metal centers upon pulsing. The new photoreductant has a large quantum yield, allowing the kinetics data to be acquired in a single flash. The net charge of +4 on Ru2Z allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. The photoexcited state Ru(II*) of Ru2Z is reduced to Ru(I) by the sacrificial electron donor aniline, and Ru(I) then reduces CuA with yields up to 60%. A stopped-flow-flash technique was used to form the pulsed state of cytochrome oxidase (the "OH" state) from several sources (bovine heart mitochondria, Rhodobacter sphaeroides, and Paracoccus denitrificans). Upon mixing the fully reduced anaerobic enzyme with oxygenated buffer containing Ru2Z, the oxidized OH state was formed within 5 ms. Ru2Z was then excited with a laser flash to inject one electron into CuA. Electron transfer from CuA --> heme a --> heme a3/CuB was monitored by optical spectroscopy, and the results were compared with the enzyme that had not been pulsed to the OH state. Pulsing had a significant effect in the case of the bovine oxidase, but this was not observed with the bacterial oxidases. Electron transfer from CuA to heme a occurred with a rate constant of 20,000 s-1 with the bovine cytochrome oxidase, regardless of whether the enzyme had been pulsed. However, electron transfer from heme a to the heme a3/CuB center in the pulsed form was 63% complete and occurred with biphasic kinetics with rate constants of 750 s-1 and 110 s-1 and relative amplitudes of 25% and 75%. In contrast, one-electron injection into the nonpulsed O form of the bovine oxidase was only 30% complete and occurred with monophasic kinetics with a rate constant of 90 s-1. This is the first indication of a difference between the fast form of the bovine oxidase and the pulsed OH form. No reduction of heme a3 is observed, indicating that CuB is the initial electron acceptor in the one-electron reduced pulsed bovine oxidase.     

Proton and electron transfer during the reduction of molecular oxygen by fully reduced cytochrome c oxidase: a flow-flash investigation using optical multichannel detection

Proton and electron transfer events during the reaction of solubilized fully reduced bovine heart cytochrome c oxidase with molecular oxygen were investigated using the flow-flash technique. Time-resolved spectral changes resulting from ligand binding and electron transfer events were detected simultaneously with pH changes in the bulk. The kinetics and spectral changes in the visible region (450-750 nm) were probed by optical multichannel detection, allowing high spectral resolution on time scales from 50 ns to 50 ms. Experiments were carried out in the presence and absence of pH-sensitive dyes (carboxyfluorescein at pH 6.5, phenol red at pH 7.5, and m-cresol purple at pH 8.5) which permitted separation of spectral changes due to proton transfer from those caused by ligand binding and electron transfer. The transient spectra recorded in the absence of dye were analyzed by singular-value decomposition and multiexponential fitting. Five apparent lifetimes (0.93 microseconds, 10 microseconds, 36 microseconds, 90 microseconds, and 1.3 ms at pH 7.5) could consistently be distinguished and provided a basis for a reaction mechanism consistent with our most recent kinetic model [Sucheta, A., Szundi, I., and Einarsdóttir, O. (1999) Biochemistry 37, 17905-17914]. The dye response indicated that proton uptake occurred concurrently with the two slowest electron transfer steps, in agreement with previous results based on single-wavelength detection [Hallén, S., and Nilsson, T. (1992) Biochemistry 31, 11853-11859]. The stoichiometry of the proton uptake reactions was approximately 1.3 +/- 0.3, 1.4 +/- 0.3, and 1.6 +/- 0.5 protons per enzyme at pH 6.5, 7.5, and 8.5, respectively. The electron transfer between heme a and CuA was limited by proton uptake on a 90 microseconds time scale. We have established the lower limit of the true rate constant for the electron transfer between CuA and heme a to be approximately 2 x 10(5) s-1.     

Time dependence of the catalytic intermediates in cytochrome c oxidase

Cytochrome c oxidase, the terminal enzyme in the electron transfer chain, catalyzes the reduction of oxygen to water in a multiple step process by utilizing four electrons from cytochrome c. To study the reaction mechanism, the resonance Raman spectra of the intermediate states were measured during single turnover of the enzyme after catalytic initiation by photolysis of CO from the fully reduced CO-bound enzyme. By measuring the change in intensity of lines associated with heme a, the electron transfer steps were determined and found to be biphasic with apparent rate constants of approximately 40 x 10(3) s(-1) and approximately 1 x 10(3) s(-1). The time dependence for the oxidation of heme a and for the measured formation and decay of the oxy, the ferryl ("F"), and the hydroxy intermediates could be simulated by a simple reaction scheme. In this scheme, the presence of the "peroxy" ("P") intermediate does not build up a sufficient population to be detected because its decay rate is too fast in buffered H(2)O at neutral pH. A comparison of the change in the spin equilibrium with the formation of the hydroxy intermediate demonstrates that this intermediate is high spin. We also confirm the presence of an oxygen isotope-sensitive line at 355 cm(-1), detectable in the spectrum from 130 to 980 micros, coincident with the presence of the F intermediate.     

Protein conformational perturbations affect the photoreduction of native cytochrome c peroxidase (III) at alkaline pH.

Ferric cytochrome c peroxidase (CCP) undergoes a ligation-state transition from a pentacoordinate, high-spin (5c/hs) heme to a hexacoordinate, low-spin (6c/1s) heme when titrated over a pH range of 7.30-9.70. This behavior is similar to that exhibited by the ferrous form of the enzyme. However, the photodissociation of the low-spin, axial ligand, exhibited by ferrous CCP at alkaline pH, is not observed for ferric CCP. Instead, a photoinduced reduction of the ferric heme is apparent in the pH range 7.90-9.70. In the absence of O2 and redox mediators such as methyl viologen (MV2+), the reoxidation of the photoreduced enzyme is very slow (tau 1/2 approximately 3 min). F(-)-bound CCP(III) (6c/hs) displays similar pH-dependent photoreduction. Horseradish peroxidase, however, does not. The formation of 6c/1s heme coincides with the onset of appreciable photoreduction (between laser pulses, > 60 ms) of CCP (III) at alkaline pH, suggesting a global protein conformational rearrangement within or around its heme pocket. Photoreduction of alkaline CCP(III) most likely involves intramolecular electron transfer (ET) from the aromatic residue in the proximal heme pocket to the photoexcited heme. We speculate that the kinetics of electron transfer are affected by changes in the orientation of Trp-191.     

Low-spin heme b(3) in the catalytic center of nitric oxide reductase from Pseudomonas nautica

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mo?ssbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mo?ssbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ～ 6 and g ～ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ～ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.     

Independent control of respiration in cytochrome c oxidase vesicles by pH and electrical gradients

The effects of altering the pH and electrical components of the membrane potential on the visible spectra and oxygen consumption rates of cytochrome oxidase vesicles were examined during steady-state respiration using cytochrome c as the substrate. Heme a was found to be 30-55% reduced in the presence of a membrane potential, becoming more reduced when the electrical gradient (delta psi) was abolished by valinomycin and more oxidized when the pH gradient (delta pH) was abolished by nigericin, with little increase (1.2-1.8-fold) in the rates of oxygen consumption in either case. When both gradients were eliminated, heme a reduction was close to initial levels, and activity was stimulated up to 8-fold. The magnitude of the changes in heme a reduction levels upon elimination of a gradient component was shown to be positively correlated with the magnitude of the respiratory control ratio of the vesicle preparation. Kinetic analysis of the dependence of oxidase activity on cytochrome c concentration indicated that changes in the Michaelis constant of the enzyme for its substrate are not a major factor in regulation by either delta pH or delta psi. These results suggest a dual mechanism for respiratory control in cytochrome oxidase vesicles under steady-state conditions, in which the electrical gradient predominantly affects electron transfer from cytochrome c to heme a, possibly by altering the reduction potential of heme a, while the pH gradient affects electron transfer from heme a (CuA) to heme a3 (CuB), possibly by a conformationally mediated change in the reduction potential of heme a3 or in the kinetics of the electron-transfer process.     

Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase

Cytochrome c oxidase couples electron transfer from cytochrome c to O 2 to proton pumping across the membrane. In the initial part of the reaction of the reduced cytochrome c oxidase with O 2, an electron is transferred from heme a to the catalytic site, parallel to the membrane surface. Even though this electron transfer is not linked to proton uptake from solution, recently Belevich et al. [(2006) Nature 440, 829] showed that it is linked to transfer of charge perpendicular to the membrane surface (electrogenic reaction). This electrogenic reaction was attributed to internal transfer of a proton from Glu286, in the D proton pathway, to an unidentified protonatable site "above" the heme groups. The proton transfer was proposed to initiate the sequence of events leading to proton pumping. In this study, we have investigated electrogenic reactions in structural variants of cytochrome c oxidase in which residues in the second, K proton pathway of cytochrome c oxidase were modified. The results indicate that the electrogenic reaction linked to electron transfer to the catalytic site originates from charge transfer within the K pathway, which presumably facilitates reduction of the site.