David F. Blair

The proton-pumping site of cytochrome c oxidase: a model of its structure and mechanism

Cytochrome c oxidase is an electron-transfer driven proton pump. In this paper, we propose a complete chemical mechanism for the enzymes proton-pumping site. The mechanism achieves pumping with chemical reaction steps localized at a redox center within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes previously determined (Blair, D.F., Gelles, J. and Chan, S.I. (1986) Biophys. J. 50, 713-733) to be needed to accomplish redox-linked proton pumping. These processes include: (1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; (2) control of the rates of the pumps redox reactions with its electron-transfer partners during the turnover cycle (gating of electrons); and (3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and sufficiently specific that a realistic assessment can be made of how well the model pump would function as a redox-linked free-energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the model would function as an efficient pump and that its behavior would be very similar to that observed of cytochrome oxidase both in the mitochondrion and in purified preparations. The analysis presented here leads to the following important general conclusions regarding the mechanistic features of the oxidase proton pump. (1) A workable proton-pump mechanism does not require large protein conformational changes. (2) A redox-linked proton pump need not display a pH-dependent midpoint potential, as has frequently been assumed. (3) Mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping.

Redox-linked proton translocation in cytochrome oxidase: the importance of gating electron flow. The effects of slip in a model transducer

In at least one component of the mitochondrial respiratory chain, cytochrome c oxidase, exothermic electron transfer reactions are used to drive vectorial proton transport against an electrochemical hydrogen ion gradient across the mitochondrial inner membrane. The role of the gating of electrons (the regulation of the rates of electron transfer into and out of the proton transport site) in this coupling between electron transfer and proton pumping has been explored. The approach involves the solution of the steady-state rate equations pertinent to proton pump models which include, to various degrees, the uncoupled (i.e., not linked to proton pumping) electron transfer processes which are likely to occur in any real electron transfer-driven proton pump. This analysis furnishes a quantitative framework for examining the effects of variations in proton binding site pKas and metal center reduction potentials, the relationship between energy conservation efficiency and turnover rate, the conditions for maximum power output or minimum heat production, and required efficiency of the gating of electrons. Some novel conclusions emerge from the analysis, including: An efficient electron transfer-driven proton pump need not exhibit a pH-dependent reduction potential; Very efficient gating of electrons is required for efficient electron transfer driven proton pumping, especially when a reasonable correlation of electron transfer rate and electron transfer exoergonicity is assumed; and A consideration of the importance and possible mechanisms of the gating of electrons suggests that efficient proton pumping by CuA in cytochrome oxidase could, in principle, take place with structural changes confined to the immediate vicinity of the copper ion, while proton pumping by Fea would probably require conformational coupling between the iron and more remote structures in the enzyme. The conclusions are discussed with reference to proton pumping by cytochrome c oxidase, and some possible implications for oxidative phosphorylation are noted.

Cyro-vibrational spectroscopy of blue copper proteins

Abstract Resonance Raman (RR) and Fourier transform infrared (FTIR) spectra at 12K have been obtained for Pseudomonas aeruginosa azurin, spinach plastocyanin, stellacyanin, and tree laccase. The temperature dependence of the azurin, plastocyanin, and stellacyanin spectra have been recorded as have the RR excitation profiles at 12 K. Room temperature RR spectra have been obtained for azurins from Alcaligenes fecalis, Alcaligenes sp., Bortadella pretussis and Bortadella bronchiseptica; bean plastocyanin; fungal laccase, human ceruloplasmin; and zucchini squash ascorbate squash ascorbate oxidase. Isotope studies employing 63 Cu/ 65 Cu and H/D substitution have been performed on the azurins from Ps. aeruginosa, Alc. fecalis , and Alc. sp . Principal conclusion include the following; The intense RR modes near 400 cm −1 include internal ligand deformations and the Cu-S(cys) stretch, rather than the Cu-S(cys) stretch and Cu-N(his-Im) stretches as previously supposed. The Cu-N(his-Im) stretches are assignable to the ubiquitous feature near 265 cm −1 ,consistent with the frequencies of similar motions in other proteins and in model complexes. Spinach plastocyanin exhibits a frequency shift of 14 cm −1 in its cysteine CS stretching frequency ( ca. 750 cm −1 ) upon freezing of the protein solution, suggesting that extra-protein forces (e.g.,solvent structure, crystallization, or substrate binding.) can influence the conformation of the active site. Above the freezing point of the solvent the plastocyanin RR modes are unusually broad, suggesting either extremely facile due to thermally accessible conformations in the high-temperature form of the active site. No selective enhancement of either the strong or weak RR modes is observed in the S(cys)→ Cu charge transfer excitation profiles of azurin, plastocyanin, or stellacyanin at 12 K. The azurin species other than Ps. aeruginosa exhibit an ‘extra» strong RR peak near 400 cm −1 which is, however, seen to be related to an unresolved shoulder in the 12 K Ps. aeruginosa spectrum. It is therefore unnecessary to invoke higher coordination numbers than four for copper to explain the RR spectra of the azurins other than Ps. aeruginosa . RR peaks appear in the azurin spectrum below 200 K which may be due to methionine C-S stretching modes and Cu-S(met) stretch or methionine C-S-C angle bend. If these low-temperature features are indeed due to such motions,methionine must be closely coordinated to copper in azurin at low temperature. The RR spectra are consistent with a monotonic relationship between the force constant of the Cu-S(cys)bond and the energy of the ligand-field transitions of the various proteins. The isotope studies lead to tentative identification of the RR modes which contain significant contributions from M-L stretches and internal histidine motions. The results provide an initial basis for reliable structural interpretation of the RR spectra of the blue copper proteins.

The metal centers of cytochrome c oxidase: Structure and function

Abstract Considerable progress has been made in recent years on the structure of the metal centers in cytochrome c oxidase. Most of these studies have naturally focused on the ligands of the metal center as they play a prominent role in electron transfer, oxygen reduction and possibly also energy conservation. The most unambiguous structural information on the ligands has emerged from EPR/ENDOR studies, particularly when these studies are undertaken in conjunction with isotopically substituted cytochrome c oxidases prepared by incorporating selectively isotopically substituted amino acids into the protein via biosynthetic procedures. These results will be reviewed. Good progress has also been made towards elucidating the mechanism of dioxygen reduction. Preliminary evidence for a mechanism involving both a peroxo and a ferryl intermediate will be presented. The possible structural differences at the dioxygen reduction site between the resting oxidized enzyme and the pulsed enzyme will also be discussed. While impressive progress has been made toward understanding the structure of the metal centers of cytochrome oxidase and their roles in dioxygen reduction, comparatively little is known about the mechanisms by which the free energy of this reaction is conserved in the form of a transmembrane electrochemical potential gradient. It appears likely that the protons consumed in the reduction of dioxygen to water are derived from the matrix side of the mitochondrial membrane. Since the electrons used in this reaction originate from the intermembrane space of the mitochondrion, a transmembrane electrochemical potential gradient will be generated. However, the spatial dispositions of the metal centers in the membrane profile (or along the transmembrane electrochemical potential profile) are not sufficiently known to allow one to pinpoint the electron transfer step(s) which will lead to energy conservation by this mechanism. The available data which bear upon this question will be discussed briefly and new experimental approaches will be suggested. Another means of conserving the available energy is pumping protons from the mitochondrial matrix to the intermembrane space. Evidence that the enzyme is, in fact, a proton pump will be reviewed, as will some of the current models for the mechanism of this pumping. The relative merits of each of the metal centers as the site of proton pumping will be considered. The implications of electron transfer rate theory for the mechanism of proton pumping and the positioning of the metal sites in the transmembrane electrochemical potential profile will be explored. It is suggested that CuA in the most suitable candidate for the proton pump; available evidence for this hypothesis will be presented. The cytochrome oxidase-catalyzed dioxygen reduction reaction will probably not release energy in uniform increments. Thus, it might be expected that some elctron-transfer steps will not lead to proton pumping, particularly under conditions of high membrane potential. This possibility has not been adequately appreciated by investigators who attempt to assign fixed proton/electron stoichiometrics to the cytochrome oxidase reaction. This question will be discussed with reference to available information on the thermodynamics of this reaction. Simple mechanisms by which the enzyme might adapt to a changing membrane potential will be described.