Accessibility of the cytochrome a heme in cytochrome c oxidase to exchangeable protons

The comparison of the resonance Raman spectrum of cytochrome a2+ from cytochrome oxidase in deuterated buffers to that in protonated buffers reveals many lines that have different frequency or intensity. Some of the frequency differences are very large, e.g. on the order of 10 cm-1. From these differences in the Raman spectra, we infer that the heme pocket is readily accessible to protons and that labile groups are either on the heme or interact strongly with it. These data suggest the possibility of direct participation in proton translocation and/or oxygen protonation by the heme of cytochrome a.     

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⁺.     

Vibrational structure of the formyl group on heme a. Implications on the properties of cytochrome c oxidase.

Resonance Raman spectra have been recorded for heme a derivatives in which the oxygen atom of the formyl group has been isotopically labeled and for Schiff base derivatives of heme a in which the Schiff base nitrogen has been isotopically labeled. The 14N-15N isotope shift in the C = N stretching mode of the Schiff base is close to the theoretically predicted shift for an isolated C = N group for both the ferric and ferrous oxidation states and in both aqueous and nonaqueous solutions. In contrast, the 16O-18O isotope shift of the C = O stretching mode of the formyl group is significantly smaller than that predicted for an isolated C = O group and is also dependent on whether the environment is aqueous or nonaqueous. This differences between the theoretically predicted shifts and the observed shifts are attributed to coupling of the C = O stretching mode to as yet unidentified modes of the heme. The complex behavior of the C = O stretching vibration precludes the possibility of making simple interpretations of frequency shifts of this mode in cytochrome c oxidase.     

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.     

Photoreactivation of the cytochrome oxidase complex with cyanide: the reaction of heme a3 photoreduction.

Electron transfer activity of isolated cytochrome oxidase inhibited by low concentrations of cyanide by 93-95% was shown to rise no less than three times under exposure to visible light. Irradiation with visible light was found to increase the rate of reduction of cytochrome oxidase heme groups in the presence of sodium dithionite. Based on these results, it is suggested that the modification of the catalytic and spectral characteristic of the cytochrome oxidase-cyanide complex is due to the photostimulation of the intramolecular electron transport at the interheme (heme a heme a3) transfer stage, i.e., is caused by photoreduction of the enzyme's heme a3-CN complex.     

The optical properties of CuA in bovine cytochrome c oxidase determined by low-temperature magnetic-circular-dichroism spectroscopy.

The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.     

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.     

Symmetry, orientation and rotational mobility in the a3 heme of cytochrome c oxidase in the inner membrane of mitochondria.

The photoinduced linear dichroism of absorption changes resulting from photolysis of the complex between heme a3 of the cytochrome oxidase and CO is studied. The experiments started from isotropic solutions or suspensions of the enzyme both in its isolated form and in mitochondria. The anisotropy responsible for the linear dichroism was induced by excitation with a flash of linearly polarized light. The dichroic ratios observed with various systems; polymerized enzyme in solution, enzyme in mitochondria and in submitochondrial particles (at 20 degrees C as well as at liquid N2-temperature) all approached a value of 4/3 which characterizes a chromophore which is circularly degenerate. Therefrom we conclude that the interaction of heme a3 with its microenvironment within the protein does not break its four-fold symmetry. The experiments with mitochondria and submitochondrial particles suspended in aqueous buffer revealed similarly high dichoric ratios without any dichroic relaxation other than a rather slow one which could be attributed to the rotation of the whole organelle in the suspending medium. Therefrom we conclude that the cytochrome oxidase either is totally immobilized in the membrane, or that it carries out only limited rotational diffusion around a single axis coinciding with the symmetry axis of heme a3. In the light of independent evidence for a transmembrane arrangement of the oxidase and for the general fluidity of the inner mitochondrial membrane we consider anisotropic mobility of the cytochrome oxidase around an axis normal to the plane of the membrane as the most likely interpretation. Then our experimental results imply that the plane of heme a3 is coplanar to the membrane.     

Isolation of a heme binding subunit from bovine heart cytochrome C oxidase.

A subunit which retains heme has been isolated and purified up to a homogenous form on polyacrylamide gel electrophoretic column in the presence of sodium dodecyl sulfate and β-mercaptoethanol from cytochrome oxidase. The separation of the subunit does not rely on any detergent except cholate used in the preparation of cytochrome oxidase. The purification involves a reaction with pyridine, pH precipitation, and DEAE-cellulose column chromatography. The purified subunit has a molecular weight of 11,600 daltons and contains more than 40 nmol Fe per mg protein; the lower iron content than the calculated value is apparently due to the loss of heme $\text{a}$ in the course of the purification. The subunit is freely soluble in aqueous solution at neutral pH to give a dark green color. Spectral properties and amino acid composition of this subunit have been studied.     

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.