Evaluating the roles of the heme a side chains in cytochrome c oxidase using designed heme proteins

Heme a is a redox cofactor unique to cytochrome c oxidases and vital to aerobic respiration. Heme a differs from the more common heme b by two chemical modifications, the C-8 formyl group and the C-2 hydroxyethylfarnesyl group. The effects of these porphyrin substituents on ferric and ferrous heme binding and electrochemistry were evaluated in a designed heme protein maquette. The maquette scaffold chosen, [Delta7-H3m](2), is a four-alpha-helix bundle that contains two bis(3-methyl-l-histidine) heme binding sites with known absolute ferric and ferrous heme b affinities. Hemes b, o, o+16, and heme a, those involved in the biosynthesis of heme a, were incorporated into the bis(3-methyl-l-histidine) heme binding sites in [Delta7-H3m](2). Spectroscopic analyses indicate that 2 equiv of each heme binds to [Delta7-H3m](2), as designed. Equilibrium binding studies of the hemes with the maquette demonstrate the tight affinity for hemes containing the C-2 hydroxyethylfarnesyl group in both the ferric and ferrous forms. Coupled with the measured equilibrium midpoint potentials, the data indicate that the hydroxyethylfarnesyl group stabilizes the binding of both ferrous and ferric heme by at least 6.3 kcal/mol via hydrophobic interactions. The data also demonstrate that the incorporation of the C-8 formyl substituent in heme a results in a 179 mV, or 4.1 kcal/mol, positive shift in the heme reduction potential relative to heme o due to the destabilization of ferric heme binding relative to ferrous heme binding. The two substituents appear to counterbalance each other to provide for tighter heme a affinity relative to heme b in both the ferrous and ferric forms by at least 6.3 and 2.1 kcal/mol, respectively. These results also provide a rationale for the reaction sequence observed in the biosynthesis of heme a.     

The cytochrome c oxidase of Paracoccus denitrificans pumps protons in a reconstituted system

The purified two-subunit cytochrome c oxidase of Paracoccus denitrificans was reconstituted into phospholipid vesicles having a high internal buffering capacity and exhibiting a respiratory control index greater than 6.6. With these proteoliposomes, pH changes of the suspending medium were monitored in response to reductant pulses in the presence of valinomycin and potassium. When reduced cytochrome c was added to allow for a limited number of turnovers (2-12), a net acidification of the extravesicular space could be observed. This apparent proton ejection by the vesicles was abolished by inhibition of the oxidase with azide, by bypassing the oxidase with ferricyanide, or by preventing charge compensation by omitting valinomycin. Addition of uncoupler led to an alkalinization, rather than an acidification, of the extravesicular space in response to reduced cytochrome c. We thus conclude that cytochrome c oxidase of P. denitrificans is a proton pump. Under the conditions described here, an apparent stoichiometry of 0.6 proton ejected/electron was obtained by extrapolation to zero turnovers.     

Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a₃, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa₃-type cytochrome c oxidase consists of overlapping bands of the hemes a²⁺ and a₃²⁺. It shows a peak at ～444 nm and a distinct shoulder at ～425 nm. However, attribution of individual spectral lineshapes to hemes a²⁺ and a₃²⁺ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a²⁺ and a₃²⁺ using two approaches. First, we reconstructed bovine CcO heme a²⁺ spectrum using a selective Ca²⁺-induced spectral shift of the heme a²⁺. Second, we investigated photobleaching of the reduced Thermus thermophilus ba₃- and bovine aa₃-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B_0x-, B_0y-transitions of both reduced hemes. The heme a²⁺ spectrum is shifted to the red relative to heme a₃²⁺ spectrum. The ～425 nm shoulder is mostly attributed to heme a₃²⁺.     

Network analysis of a proposed exit pathway for protons to the P-side of cytochrome c oxidase

Cytochrome c Oxidase (CcO) reduces O₂, the terminal electron acceptor, to water in the aerobic, respiratory electron transport chain. The energy released by O₂ reductions is stored by removing eight protons from the high pH, N-side, of the membrane with four used for chemistry in the active site and four pumped to the low pH, P-side. The proton transfers must occur along controllable proton pathways that prevent energy dissipating movement towards the N-side. The CcO N-side has well established D- and K-channels to deliver protons to the protein interior. The P-side has a buried core of hydrogen-bonded protonatable residues designated the Proton Loading Site cluster (PLS cluster) and many protonatable residues on the P-side surface, providing no obvious unique exit. Hydrogen bond pathways were identified in Molecular Dynamics (MD) trajectories of Rb. sphaeroides CcO prepared in the P_R state with the heme a₃ propionate and Glu286 in different protonation states. Grand Canonical Monte Carlo sampling of water locations, polar proton positions and residue protonation states in trajectory snapshots identify a limited number of water mediated, proton paths from PLS cluster to the surface via a (P-exit) cluster of residues. Key P-exit residues include His93, Ser168, Thr100 and Asn96. The hydrogen bonds between PLS cluster and P-exit clusters are mediated by a water wire in a cavity centered near Thr100, whose hydration can be interrupted by a hydrophobic pair, Leu255B (near Cu_A) and Ile99. Connections between the D channel and PLS via Glu286 are controlled by a second, variably hydrated cavity.

Resonance Raman evidence for an exchangeable protein hydrogen associated with the heme a group of cytochrome oxidase

When cytochrome-c oxidase is soaked in D2O, downshifts of the cytochrome a formyl C = O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D2O-induced RR changes are fully developed during 3-5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H-bonds to the heme formyl. The rate of the D2O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H-bond is directly involved in the redox-linked mechanism of proton pumping.     

Probing the access of protons to the K pathway in the Paracoccus denitrificans cytochrome c oxidase

In recent studies on heme-copper oxidases a particular glutamate residue in subunit II has been suggested to constitute the entry point of the so-called K pathway. In contrast, mutations of this residue (E78(II)) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78(II)Q) or reduce it to about 50% (E78(II)A); in the latter case, the mutation causes no drastic decrease in heme a(3) reduction kinetics under anaerobic conditions, when compared to typical K pathway mutants. Moreover, both mutant enzymes retain full proton-pumping competence. While oxidized-minus-reduced Fourier-transform infrared difference spectroscopy demonstrates that E78(II) is indeed addressed by the redox state of the enzyme, absence of variations in the spectral range characteristic for protonated aspartic and glutamic acids at approximately 1760 to 1710 cm(-1) excludes the protonation of E78(II) in the course of the redox reaction in the studied pH range, although shifts of vibrational modes at 1570 and 1400 cm(-1) reflect the reorganization of its deprotonated side chain at pH values greater than 4.8. We therefore conclude that protons do not enter the K channel via E78(II) in the Paracoccus enzyme.     

Localization of Cu and heme a on cytochrome c oxidase polypeptides.

After mild dissociation of cytochrome $\text{c}$ oxidase protomers, and polyacrylamide gel electrophoresis, copper was found predominantly in polypeptides of Bands V (m.w. 12,100) and VII (m.w. 3,400), and heme a predominantly in polypeptides of Bands I (m.w. 35,300) and II (m.w. 21,000). Some copper was found in Band II – III, and heme a in Band V.     

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.     

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.     

The protonation state of a heme propionate controls electron transfer in cytochrome c oxidase

In cytochrome c oxidase (CcO), exergonic electron transfer reactions from cytochrome c to oxygen drive proton pumping across the membrane. Elucidation of the proton pumping mechanism requires identification of the molecular components involved in the proton transfer reactions and investigation of the coupling between internal electron and proton transfer reactions in CcO. While the proton-input trajectory in CcO is relatively well characterized, the components of the output pathway have not been identified in detail. In this study, we have investigated the pH dependence of electron transfer reactions that are linked to proton translocation in a structural variant of CcO in which Arg481, which interacts with the heme D-ring propionates in a proposed proton output pathway, was replaced with Lys (RK481 CcO). The results show that in RK481 CcO the midpoint potentials of hemes a and a(3) were lowered by approximately 40 and approximately 15 mV, respectively, which stabilizes the reduced state of Cu(A) during reaction of the reduced CcO with O(2). In addition, while the pH dependence of the F --> O rate in wild-type CcO is determined by the protonation state of two protonatable groups with pK(a) values of 6.3 and 9.4, only the high-pK(a) group influences this rate in RK481 CcO. The results indicate that the protonation state of the Arg481 heme a(3) D-ring propionate cluster having a pK(a) of approximately 6.3 modulates the rate of internal electron transfer and may act as an acceptor of pumped protons.     

Self-assembly of heme A and heme B in a designed four-helix bundle: implications for a cytochrome c oxidase maquette

Heme A, a prosthetic group of cytochrome c oxidase [EC 1.9.3.1], has been introduced into two de novo designed four helix bundle proteins, [H10A24](2) and [H10H24](2), known to bind 2-4 equiv of heme B, respectively [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A., J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425-432]. [H10A24](2), [Ac-CGGGELWKL x HEELLKK x FEELLKL x AEERLKK x L-CONH(2)](2)(2), binds two heme A molecules per four-helix unit via bis-histidine ligation at the 10,10' positions with measured K(d) values of <0.1 and 5 nM, values much lower than those measured for heme B (K(d) values of 50 and 800 nM). The heme A-protein complex, [heme A-H10A24](2), exhibits well-defined absorption spectra in both the ferric and ferrous states, and an electron paramagnetic resonance spectrum characteristic of a low spin heme in the ferric form. A single midpoint redox potential (E(m8)) was determined for [heme A-H10A24](2) at -45 mV (vs SHE), which is significantly higher than that of the protein bound heme B (-130 and -200 mV). The observation of a single midpoint redox potential for [heme A-H10A24](2) and a pair of midpoints for [heme B-H10A24](2) indicates that the di-alpha-helical monomers are oriented in an anti topology (disulfides on opposite sides of bundle) in the former (lacking heme-heme electrostatic interaction) and syn in the latter. A mixture of global topologies was indicated by the potentiometric titration of the related [heme A-H10H24](2) which possess two distinct reduction potentials of +41 (31%) and -65 mV (69%). Self-assembly of the mixed cofactor heme A-heme B-[H10A24](2) was accomplished by addition of a single equivalent of each heme A and heme B to [H10A24](2). The single midpoint redox potential of heme B, E(m8) = -200 mV, together with the split midpoint redox potential of heme A in heme A-heme B-[H10A24](2), E(m8) = +28 mV (33%) and -65 mV (67%), indicated the existence of both syn and anti topologies of the two di-alpha-helical monomers in this four helix bundle. Synthesis of the mixed cofactor [heme A-heme B-H10H24](2) was accomplished by addition of a 2 equiv of each heme A and heme B to [H10H24](2) and potentiometry indicated the pair of hemes B resided in the 10,10' sites and heme A occupied the 24,24' sites. The results indicate that heme peripheral structure controls the orientation of the di-alpha-helical monomers in the four-helix bundle which are interchangeable between syn and anti topologies. In the reduced form, [heme A-H10A24](2), reacts quantitatively to form [carbonmonoxy-heme A-H10A24](2) as evidenced by optical spectroscopy. The synthetic [heme A-H10A24](2) can be enzymatically reduced by NAD(P)H with natural reductases under anaerobic conditions, and reversibly oxidized by dioxygen to the ferric form.     

Nuclear magnetic resonance study of exchangeable protons in ferrocytochrome c

Five well-resolved exchangeable proton resonances have been observed in horse ferrocytochrome c in the low-field region between ?10.0 and ?12.0 ppm. A resonance at ?11.6 ppm is assigned to an amino proton of His 26 on the basis of its magnetic field position, pH dependence and its correlation with histidine at this position in eight species. A resonance at ?10.9 ppm observed in horse and donkey ferrocytochrome c is assigned to a hydrogen-bonded ?-amino proton of Lys 60. This resonance shifts upfield with increasing salt and decreasing pH, with the shifts increasing as one goes from chloride to bromide to iodide. With less assurance, a resonance at ?10.6 ppm is assigned to the indole amino of Trp 59 and a resonance at ?10.3 ppm to the amino proton of the His 18. These resonances have been used to study the binding of small anions to ferrocytochrome c, the results of which show that inorganic phosphate, ADP and ATP all bind in the immediate vicinity of His 26, and that the inorganic phosphate has the greatest effect upon the imidazole amino exchange time.     

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase.

Dilatometry is a sensitive technique for measuring volume changes occurring during a chemical reaction. We applied it to the reduction-oxidation cycle of cytochrome c oxidase, and to the binding of cytochrome c to the oxidase. We measured the volume changes that occur during the interconversion of oxidase intermediates. The numerical values of these volume changes have allowed the construction of a thermodynamic cycle that includes many of the redox intermediates. The system volume for each of the intermediates is different. We suggest that these differences arise by two mechanisms that are not mutually exclusive: intermediates in the catalytic cycle could be hydrated to different extents, and/or small voids in the protein could open and close. Based on our experience with osmotic stress, we believe that at least a portion of the volume changes represent the obligatory movement of solvent into and out of the oxidase during the combined electron and proton transfer process. The volume changes associated with the binding of cytochrome c to cytochrome c oxidase have been studied as a function of the redox state of the two proteins. The volume changes determined by dilatometry are large and negative. The data indicate quite clearly that there are structural alterations in the two proteins that occur on complex formation.     

The identification of histidine ligands to cytochrome a in cytochrome c oxidase

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a.     

Reconstruction of absolute absorption spectrum of reduced heme a in cytochrome c oxidase from bovine heart

This paper presents a new experimental approach for determining the individual optical characteristics of reduced heme a in bovine heart cytochrome c oxidase starting from a small selective shift of the heme a absorption spectrum induced by calcium ions. The difference spectrum induced by Ca²⁺ corresponds actually to a first derivative (differential) of the heme a ²⁺ absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a ²⁺ a ₃ ³⁺ -CN) and was subsequently used as a basis spectrum for further procession and modeling. The individual absorption spectrum of the reduced heme a in the Soret region was reconstructed as the integral of the difference spectrum induced by addition of Ca²⁺. The spectrum of heme a ²⁺ in the Soret region obtained in this way is characterized by a peak with a maximum at 447 nm and half-width of 17 nm and can be decomposed into two Gaussians with maxima at 442 and 451 nm and half-widths of ～10 nm (589 cm^?1) corresponding to the perpendicularly oriented electronic π→π* transitions B _0x and B _0y in the porphyrin ring. The reconstructed spectrum in the Soret band differs significantly from the “classical” absorption spectrum of heme a ²⁺ originally described by Vanneste (Vanneste, W. H. (1966) Biochemistry, 65, 838–848). The differences indicate that the overall γ-band of heme a ²⁺ in cytochrome oxidase contains in addition to the B _0x and B _0y transitions extra components that are not sensitive to calcium ions, or, alternatively, that the Vanneste’s spectrum of heme a ²⁺ contains significant contribution from heme a ₃ ²⁺ . The reconstructed absorption band of heme a ²⁺ in the α-band with maximum at 605 nm and half-width of 18 nm (850 cm^?1) corresponds most likely to the individual Q _0y transition of heme a, whereas the Q _0x transition contributes only weakly to the spectrum.