Center Of Cytochrome Research Articles

Engineering the Electronic Structure of Active Centers in Metalloporphyrins to Boost Oxygen Reduction Reaction Activity

AbstractInspired by the ligand‐promoted high reactivity of the heme active center in cytochrome c oxidase (CcO) toward oxygen reduction reaction (ORR), this work proposes a new strategy for improving the efficiency of metalloporphyrin‐catalyzed ORR. We carried out a comparative study between two graphene/cobalt porphyrin complexes, the rGO/TMPPCo formed by noncovalent π‐π stacking, and the rGO‐Ci/TMPPCo with cobalt porphyrins (TMPPCo) axially coordinated with the 1‐carboimidazole (Ci) ligands on reduced graphene oxide (rGO) surface. The involvement of the axial ligand was found to greatly reduce the ORR overpotential by 120 mV in acidic medium, while hydrodynamic tests revealed an unaltered reaction mechanism. This positive effect of axial ligand has been rationalized by both electronic “push effect” and steric “trans effect”. The results of this work highlight the importance of a rationally engineered electronic structure of active sites in heterogeneous catalysis, providing insights for the fabrication of highly‐efficient ORR electrocatalysts.

Protonation of the oxo-bridged heme/copper assemblies: Modeling the oxidized state of the cytochrome c oxidase active site

In this study on model compounds for the resting oxidized state of the iron‑copper binuclear center in cytochrome c oxidase (CcO), we describe the synthesis of a new μ-oxo-heme/Cu complex, [(TPP)FeIII–O–CuII(tmpa)][B(C6F5)4] (2) {TPP: tetraphenyl porphyrinate(2–); TMPA: tris(2-pyridylmethylamine)}, as well as two protonation events for three μ-oxo-heme/Cu complexes with varying peripheral substituents on the heme site. The addition of increasing amounts of strong acid to these μ-oxo-heme/Cu systems successively led to the generation of the corresponding μ-hydroxo, μ-aquo, and the dissociated complexes. The heme/Cu assemblies bridged through a water ligand are reported here for the first time and the 1H NMR and 19F NMR spectral properties are consistent with antiferromagnetically coupled high-spin iron(III) and copper(II) centers. By titration using a series of protonated amines, the pKa values for the corresponding μ-hydroxo-heme/Cu species (i.e., the first protonation event) have been reported and compared with the pKa ranges previously estimated for related systems. These synthetic systems may represent structural models for the oxidized FeIII–X–CuII resting state, or turnover intermediates and can be employed to clarify the nature of proton/electron transfer events in CcO. SynopsisThe resting oxidized state of the cytochrome c oxidase active site contains an Fea3−OHx−CuB moiety. Here, we investigated two successive protonation events, for a series of μ-oxo-heme/Cu assemblies and reported the pKa values for the first protonation event. The μ-aquo-heme/Cu complexes described here are the first examples of such systems.

Probing Protein Secondary Structure Influence on Active Centers with Hetero Two-Dimensional Correlation (Resonance) Raman Spectroscopy: A Demonstration on Cytochrome C.

The functionality of active centers in proteins is governed by the secondary and higher structure of proteins which often lead to structures in the active center that are different from the structures found in protein-free models of the active center. To elucidate this structure–function relationship, it is therefore necessary to investigate both the protein structure and the local structure of the active center. In this work, we investigate the application of hetero (resonance) Raman two-dimensional correlation spectroscopy (2D-COS) to this problem. By employing a combination of near-infrared-Fourier transform-Raman- and vis-resonance Raman spectroscopy, we could show that this combination of techniques is able to directly probe the structure–function relationship of proteins. We were able to correlate the transition of the heme center in cytochrome c from low to high spin with changes in the secondary structure with the above mentioned two spectroscopic in situ techniques and without sample preparation. Thereby, we were able to reveal that the combination of a spectroscopic method to selectively observe the active center with a technique that monitors the whole system offers a promising toolkit to investigate the structure–function relationship of proteins with photoactive centers in general.

Broken Symmetry DFT Calculations/Analysis for Oxidized and Reduced Dinuclear Center in Cytochrome c Oxidase: Relating Structures, Protonation States, Energies, and Mössbauer Properties in ba3 Thermus thermophilus.

The Fea33+···CuB2+ dinuclear center (DNC) structure of the as-isolated oxidized ba3 cytochrome c oxidase (CcO) from Thermus thermophilus (Tt) is still not fully understood. When the proteins are initially crystallized in the oxidized state, they typically become radiolyticly reduced through X-ray irradiation. Several X-ray crystal structures of reduced ba3 CcO from Tt are available. However, depending on whether the crystals were prepared in a lipidic cubic phase environment or in detergent micelles, and whether the CcO’s were chemically or radiolyticly reduced, the X-ray diffraction analysis of the crystals showed different Fea32+···CuB+ DNC structures. On the other hand, Mössbauer spectroscopic experiments on reduced and oxidized ba3 CcOs from Tt (Zimmermann et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5779–5783) revealed multiple 57Fea32+ and 57Fea33+ components. Moreover, one of the 57Fea33+ components observed at 4.2 K transformed from a proposed “low-spin” state to a different high-spin species when the temperature was increased above 190 K, whereas the other high-spin 57Fea33+ component remained unchanged. In the current Article, in order to understand the heterogeneities of the DNC in both Mössbauer spectra and X-ray crystal structures, the spin crossover of one of the 57Fea33+ components, and how the coordination and spin states of the Fea33+/2+ and Cu2+/1+ sites relate to the heterogeneity of the DNC structures, we have applied density functional OLYP calculations to the DNC clusters established based on the different X-ray crystal structures of ba3 CcO from Tt. As a result, specific oxidized and reduced DNC structures related to the observed Mössbauer spectra and to spectral changes with temperature have been proposed. Our calculations also show that, in certain intermediate states, the His233 and His283 ligand side chains may dissociate from the CuB+ site, and they may become potential proton loading sites during the catalytic cycle.

The structure of a ferrous heme-nitro species in the binuclear heme a3/CuB center of ba3-cytochrome c oxidase as determined by resonance Raman spectroscopy.

Members of the cytochrome c oxidase family exhibit nitrite reductase activity. In this work, we have characterized a ferrous heme a3-nitro species in ba3-oxidase by resonance Raman spectroscopy. This provides the first evidence for the structure of a nitrite-bound species in the binuclear heme/copper center of cytochrome c oxidases.

Mechanism of Copper Incorporation in Subunit II of Cytochrome c Oxidase from Thermus thermophilus: Identification of Intermediate Species

Detailed spectroscopic and kinetic studies of incorporation of copper ion in the wild type (WT) and the D111AA (AA = K, N, or E) mutants of the metal ion binding site of the soluble fragment of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) showed the formation of at least two distinct intermediates. The global analyses of the multiwavelength kinetic results suggested a four-step reaction scheme involving two distinct intermediates in the pathway of incorporation of copper ions into the apoprotein forming the purple dinuclear CuA. An early intermediate similar to the red copper binding proteins was detected in the WT as well as in all the mutants. The second intermediate was a green copper species in the case of WT TtCuA. Mutation of Asp111, however, formed a second intermediate that is distinctly different from that formed in the case of the WT protein, suggesting that mutants follow pathways of copper ion incorporation different from that in the WT protein. The electrostatic interaction between Asp111 and the coordinating His114 possibly plays a subtle role in the mechanism of incorporation of metal ion into the protein. The overall Kd for WT TtCuA was found to be ~8 nM, which changed with mutation of the Asp111 residue. The activation and thermodynamic parameters were also determined from the temperature- and pH-dependent multiwavelength kinetics, and the results are discussed to unravel the role of Asp111 in the mechanism of formation of the dinuclear CuA center in cytochrome c oxidase.

Detection of the hyponitrite species (HO‐N=N‐O) in denitrification: Reactivity of NO with the heme Fe‐Cu center of cytochrome caa3 and the heme Fe –Fe center of Nitric oxide reductase

Nitric oxide reductase (NOR) is the third of the four enzymes of bacterial denitrification that is responsible for the catalytic formation of laughing gas (N2O). Here we report on the formation of the hyponitrite (HO‐N=N‐O−) species (νN‐N=1332 cm−1) in the heme b3 Fe/non‐heme FeB dinuclear center of NOR from Paracoccus denitrificans. With the identification of the hyponitrite species the mechanism for the 2e−/2H+ reduction of NO to N2O can be described with more certainty.This work was financially supported by the Cyprus Research Promotion Foundation grant TEXNOLOGIA/THEPIS/0609(BE)/05.

Polymorphism of the mitochondrial cytochrome b gene, phylogeography, and molecular adaptation of the Siberian salamander (Salamandrella keyserlingii, Amphibia, Caudata)

We performed an analysis of nucleotide variation of the mitochondrial DNA (mtDNA) cytochrome b gene in populations of the Siberian salamander (Salamandrella keyserlingii Dybowski, 1870) from Khabarovsk krai; the Jewish autonomous oblast; Amur, Sakhalin, and Magadan oblasts; Sakha; and other regions of northern Eurasia. Two major phylogenetic haplotype groups, AB and C, were found using phylogenetic analysis. The degree of intergroup divergence is 1.87%, which indicates that these groups diverged about 1.5 Ma ago. Siberian salamanders with the AB haplogroup and with four of the five subgroups of the C haplogroups are found only in the Far East, including Sakhalin; the rest of the geographical range is occupied by one of the haplogroups, C1. Analysis of the distribution of nonsynonymous and synonymous substitutions demonstrated that the cytochrome b gene variation of the Siberian salamander is neutral. However, nucleotide sequences of the AB and C groups differ significantly in the region corresponding to the cd-loop of the Q0 redox center of cytochrome b protein. In addition, the radical serine-to-leucine substitution in position 160 of the cd2-loop changes the physicochemical properties of the cd region of the C group sequences by increasing its hydrophobicity. The obtained results suggest that the changes in the sequence of the cytochrome b gene of the Siberian salamander group C are adaptive.

EPR Evidence of Cyanide Binding to the Mn(Mg) Center of Cytochrome c Oxidase: Support for CuA−Mg Involvement in Proton Pumping

We examined the anion binding behavior of the Mg(Mn) site in cytochrome c oxidase to test a possible role of this center in proton pumping. Rhodobacter sphaeroides grown in a Mn(II)-rich medium replaces the intrinsic Mg(II) ion with an EPR-detectable Mn(II) ion without change in activity. Due to its close proximity and a shared ligand, oxidized Cu(A) is spin-coupled to the Mn(II) ion, affecting the EPR spectrum. An examination of both bovine and R.s. oxidase crystal structures reveals a hydrogen-bonding pattern in the vicinity of the Mg(II) site that is consistent with three water ligands of the Mg(Mn) center when Cu(A) is oxidized. In the reduced structure, one water molecule in the vicinity of the Cu(A) ligand, E198, moves closer, appearing to be converted into an ionically bonded hydronium ion, while a second water molecule bonded to Mg(Mn) shows evidence of conversion to a hydroxide. The implied proton movement is proposed to be part of a redox-linked export of a pumped proton from the binuclear center into the exit pathway. To test the model, cyanide and azide were added to the oxidized and reduced forms of the enzyme, and Mn(II) CW-EPR and ESEEM spectra were recorded. Addition of azide broadened the CW-EPR spectra for both oxidized and reduced enzyme. Cyanide addition affected the Mn(II) CW-EPR spectrum of reduced cytochrome c oxidase by increasing Mn(II) zero field splitting and broadening the spectral line shapes but had no effect on oxidized enzyme. ESEEM measurements support a differential ability of Mn(II) to bind cyanide in the reduced state of cytochrome c oxidase. This new observation of anion binding at the Mg/Mn site is of interest in terms of accessibility of the buried site and its potential role in redox-dependent proton pumping.

A New Iron-oxidizing/O2-reducing Supercomplex Spanning Both Inner and Outer Membranes, Isolated from the Extreme Acidophile Acidithiobacillus ferrooxidans

The iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans involves various metalloenzymes. Here we demonstrate that the oxygen reduction pathway from ferrous iron (named downhill pathway) is organized as a supercomplex constituted of proteins located in the outer and inner membranes as well as in the periplasm. For the first time, the outer membrane-bound cytochrome c Cyc2 was purified, and we showed that it is responsible for iron oxidation and determined that its redox potential is the highest measured to date for a cytochrome c. The organization of metalloproteins inside the supramolecular structure was specified by protein-protein interaction experiments. The isolated complex spanning the two membranes had iron oxidase as well as oxygen reductase activities, indicating functional electron transfer between the first iron electron acceptor, Cyc2, and the Cu(A) center of cytochrome c oxidase aa(3). This is the first characterization of a respirasome from an acidophilic bacterium. In Acidithiobacillus ferrooxidans,O(2) reduction from ferrous iron must be coupled to the energy-consuming reduction of NAD(+)(P) from ferrous iron (uphill pathway) required for CO(2) fixation and other anabolic processes. Besides the proteins involved in the O(2) reduction, there were additional proteins in the supercomplex, involved in uphill pathway (bc complex and cytochrome Cyc(42)), suggesting a possible physical link between these two pathways.

Protein−Protein Interactions Studied by EPR Relaxation Measurements: Cytochrome c and Cytochrome c Oxidase

The complex formed between cytochrome c oxidase from Paracoccus denitrificans and its electron-transfer partner cytochrome c has been studied by multi-frequency pulse electron paramagnetic resonance spectroscopy. The dipolar relaxation of a fast-relaxing paramagnetic center induced on a more slowly relaxing center can be used to measure their distance in the range of 1-4 nm. This method has been used here for the first time to study transient protein-protein complex formation, employing soluble fragments for both interacting species. We observed significantly enhanced transversal relaxation of the CuA center in cytochrome c oxidase due to the fast-relaxing iron of cytochrome c upon complex formation. The possibility to measure cytochrome c oxidase in the presence and absence of cytochrome c permitted us to separate the dipolar relaxation from other relaxation contributions. This allowed a quantitative simulation and interpretation of the relaxation data. The specific temperature dependence of the dipolar relaxation together with the high orientational selectivity achieved at high magnetic field values may provide detailed information on distance and relative orientation of the two proteins with respect to each other in the complex. Our experimental results cannot be explained by any single well-defined structure of the complex of cytochrome c oxidase with cytochrome c, but rather suggest that a broad distribution in distances and relative orientations between the two proteins exist within this complex.

The Two-State Issue in the Mixed-Valence Binuclear CuA Center in Cytochrome c Oxidase and N2O Reductase

For the CuA site in the protein, sigmau* and piu are the ground and lowest energy excited-states, respectively. EPR data on CuA proteins show a low g parallel value of 2.19 which derives from spin-orbital coupling between sigmau* and piu which requires an energy gap between sigmau* and piu of 3000-4500 cm-1. On the other hand, from paramagnetic NMR studies, it has been observed that the first excited-state is thermally accessible and the energy gap between the ground state and the thermally accessible state is approximately 350 cm-1. This study addressed this apparent discrepancy and evaluated the roles of the two electronic states, sigmau* and piu, in electron transfer (ET) of CuA. The potential energy surface calculations show that both NMR and EPR results are consistent with the electronic/geometric structure of CuA. The anti-Curie behavior observed in paramagnetic NMR studies of CuA results from the thermal equilibrium between the sigmau* and piu states which are at very close energies in their respective equilibrium geometries. Alternatively, the EPR g-value analysis involves the sigmau* ground state in the geometry with a short dCu-Cu where the piu state is a Frank-Condon excited-state with the energy of 3200 cm-1. The protein environment plays a role in maintaining CuA in the sigmau* state as a lowest-energy state with the lowest reorganization energy and high-covalent coupling to the Cys and His ligands for efficient intra- and intermolecular ET with a low-driving force.

ENDOR Investigation of the Liganding Environment of Mixed-Spin Ferric Cytochromec‘

The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) heme center in cytochrome c' was investigated by electron nuclear double resonance (ENDOR), a technique not previously applied to this mixed-spin system. Cytochrome c' was obtained from overexpressing variants of Rhodobacter sphaeroides 2.4.3. ENDOR for this study was done at the g(//) = 2.00 extremum where single-crystal-like, well-resolved spectra prevail. The heme meso protons of cytochrome c' showed a contact interaction that implied spin delocalization arising from the heme (d(z)(2)) orbital enhanced by iron out-of-planarity. An exchangeable proton ENDOR feature appeared from the proximal His123 Ndelta hydrogen. This Ndelta hydrogen, which crystallographically has no hydrogen-bonding partner and thus belongs to a neutral imidazole, showed a larger hyperfine coupling than the corresponding hydrogen-bonded Ndelta proton from metmyoglobin. The unique residue Phe14 occludes binding of a sixth ligand in cytochrome c', and ENDOR from a proton of the functionally important Phe14 ring, approximately 3.3 A away from the heme iron, was detected. ENDOR of the nitrogen ligand hyperfine structure is a direct probe into the sigma-antibonding (d(z)(2)) and (d(x)(2)-d(y)(2)) orbitals whose energies alter the relative stability and admixture of sextet and quartet states and whose electronic details were thus elucidated. ENDOR frequencies showed for cytochrome c' larger hyperfine couplings to the histidine nitrogen and smaller hyperfine couplings to the heme nitrogens than for high-spin ferric hemes. Both of these findings followed from the mixed-spin ground state, which has less (d(x)(2)-d(y)(2)) character than have fully high-spin ferric heme systems.

Identification of a Disulfide Switch in BsSco, a Member of the Sco Family of CytochromecOxidase Assembly Proteins,

BsSco is a membrane-associated protein from Bacillus subtilis characterized by the sequence CXXXCP, which is conserved in yeast and human mitochondrial Sco proteins, and their bacterial homologues. BsSco is involved in the assembly of the Cu(A) center in cytochrome c oxidase and may play a role in the transfer of copper to this site. We have characterized the soluble domain of BsSco by biochemical, spectroscopic, and structural approaches. Soluble BsSco is monomeric in solution, and the two conserved cysteines are involved in an intramolecular cystine bridge. The cystine bridge is easily reduced, and circular dichroism spectroscopy shows no large-scale changes in BsSco's secondary structure upon reduction. The crystal structure of soluble BsSco, determined at 1.7 A resolution, reveals typical elements of a thioredoxin fold. The CXXXCP motif, in which Cys45 and Cys49 are conserved, is located in a turn structure on the surface of the protein. In various native and His135Ala mutant structures, both disulfide-bonded and non-disulfide-bonded forms of CXXXCP are observed. However, despite extensive attempts, copper has not been found near or beyond the CXXXCP motif, a presumptive copper-binding site. Another potential copper binding residue, His135, is located in a highly flexible loop parallel to the CXXXCP loop but is more than 10 A from Cys45 and Cys49. If these three residues are to coordinate copper, a conformational change is necessary. The structural identification of a disulfide switch demonstrates that BsSco has the capability to fill a redox role in Cu(A) assembly.

Control of Mitochondrial Respiration by NO., Effects of Low Oxygen and Respiratory State

Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme a3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 microM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 +/- 0.01 microm in state 3 versus 0.31 +/- 0.04 microM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.

Redox properties of an engineered purple Cu A azurin

Purple Cu A centers are a class of binuclear, mixed-valence copper complexes found in cytochrome c oxidase and nitrous oxide reductase. An engineered Cu A protein was formed by replacing a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Pseudomonas aeruginosa azurin with the corresponding portion of sequence from the Cu A center of cytochrome c oxidase from Paracoccus denitrificans [Proc. Natl. Acad. Sci. USA 93 (1996) 461]. Oxidation–reduction midpoint potential ( E m) values of the Cu A azurin of +399±10 and +380±2 mV, respectively, were determined by cyclic voltammetry and spectrochemical titration. An n value of one was obtained, indicating that the redox reaction is cycling between the mixed valence and the fully reduced states. Whereas the E m value of native azurin is pH dependent, the E m value of Cu A azurin is not, as expected for the Cu A center. Similarities and differences in the redox properties are discussed in terms of the known crystal structures of Cu A centers in cytochrome c oxidase and Cu A azurin.

A switch in the electron transfer from heme a to binuclear centre of cytochrome c oxidase

New experimental evidence that a switch controls the reduction of the heme a3-CuB binuclear centre has been observed in the N2-dried thin film of purified cytochrome oxidase. When immersing the enzyme film into the acid phosphate buffer with extremely low concentration of dithionite, a spectrum was given to show a reduction of heme a with no electrons resting on CuA. By increasing dithionite, electrons could be accumulated gradually on CuA, but the binuclear centre still remains in the oxidized state. When the accumulation of electrons on CuA and/or heme a exceeded a threshold, a turnover of reduction of the binuclear centre and oxidation of heme a occurred abruptly. This switch-like action is pH-dependent.

The Role of the Cross-link His-Tyr in the Functional Properties of the Binuclear Center in Cytochrome c Oxidase

Resonance Raman and Fourier transform infrared spectroscopies have been used to study the aa(3)-type cytochrome c oxidase and the Y280H mutant from Paracoccus denitrificans. The stability of the binuclear center in the absence of the Tyr(280)-His(276) cross-link is not compromised since heme a(3) retains the same proximal environment, spin, and coordination state as in the wild type enzyme in both the oxidized and reduced states. We observe two C-O modes in the Y280H mutant at 1966 and 1975 cm(-1). The 1975 cm(-1) mode is assigned to a gamma-form and represents a structure of the active site in which Cu(B) exerts a steric effect on the heme a(3)-bound CO. Therefore, the role of the cross-link is to fix Cu(B) in a certain configuration and distance from heme a(3), and not to allow histidine ligands to coordinate to Cu(B) rather than to heme a(3), rendering the enzyme inactive, as proposed recently (Das, T. K., Pecoraro, C., Tomson, F. L., Gennis, R. B., and Rousseau, D. L. (1998) Biochemistry 37, 14471-14476). The results provide solid evidence that in the Y280H mutant the catalytic site retains its active configuration that allows O(2) binding to heme a(3). Oxygenated intermediates are formed by mixing oxygen with the CO-bound mixed-valence wild type and Y280H enzymes with similar Soret maxima at 438 nm.

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: I. Spectral and functional properties.

To study the functional significance of the unusual bimetallic Cu(A) center of cytochrome c oxidase, the direct ligands of the Cu(A) center in subunit II of the holoenzyme were mutated. Two of the mutant forms, M263L and H260N, exhibit major changes in activity (10% and 1% of wild-type, respectively) and in near-infrared and EPR spectra, but metal analysis shows that both mutants retain two coppers in the Cu(A) center and both retain proton pumping activity. In M263L, multifrequency EPR studies indicate the coppers are still electronically coupled, while all the other metal centers in M263L appear unchanged, by visible, EPR, and FTIR spectroscopy. Nevertheless, heme a3 is very slow to reduce with cytochrome c or dithionite under stopped-flow and steady-state conditions. This effect appears to be secondary to the change in redox equilibrium between Cu(A) and heme a. The studies reported here and in Wang et al. [Wang, K., Geren, L., Zhen, Y., Ma, L., Ferguson-Miller, S., Durham, B., and Millett, F. (2002) Biochemistry 41, 2298-2304] demonstrate that altering the ligands of Cu(A) can influence the rate and equilibrium of electron transfer between Cu(A) and heme a, but that the native ligation state is not essential for proton pumping.

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer.

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.