Analysis of the kinetics of the membrane potential generated by cytochrome c oxidase upon single electron injection

Abstract

In a recent work from this group (Popovic, D. M.; Stuchebrukhov A. A. FEBS Lett. 2004, 566, 126), a model of proton pumping by cytochrome c oxidase (CcO) was proposed. The key element of the model is His291 (bovine notation), a histidine ligand to enzyme's CuB redox center, which plays the role of the pump element. The model assumes that upon electron transfer between heme a and the binuclear catalytic center of the enzyme, two sequential proton transfers occur: First, a proton from Glu242 is transferred to an unprotonated His291, then a second proton, after reprotonation of Glu242 from the negative side of the membrane, is transferred to a hydroxyl group in the binuclear center, a water molecule is formed, and the first proton, due to proton-proton repulsion, is expelled from His291 to the positive side of the membrane, resulting in a pumping event. In the process the free energy of water formation (i.e., reduction of oxygen) is transformed into a proton gradient across the membrane. The model possesses specific kinetic features. It assumes, for example, that upon electron transfer the first proton is transferred to the proton-loading site of the pump, His291, and not to the catalytic center of the enzyme. Here, we analyze the kinetic properties of the proposed model, and calculate the time dependence of the membrane potential generated by CcO upon a single electron injection into the enzyme. These data are directly compared with recent experimental measurements of the membrane potential generated by CcO. Specifically, F to O, and O to E transitions will be discussed. Several enzymes from different organisms (bovine, two bacterial enzymes, and several mutants) are compared and discussed in detail. The kinetic description, however, is phenomenological, and does not include explicitly the nature of the groups involved in proton translocation, except in terms of their position depth within the membrane; thus, the kinetic equations developed here are in fact describe a generic model, similar, e.g., to that proposed earlier by Peter Rich (P.R. Rich, Towards an understanding of the chemistry of oxygen reduction and proton translocation in the iron-copper respiratory oxidases. Aust. J. Plant Physiol. 22 (1995) 479-486), and which is based on the idea of displacement of the pumped protons by the chemical ones.

Partial Steps of Charge Translocation in the Nonpumping N139L Mutant of Rhodobacter sphaeroides Cytochrome c Oxidase with a Blocked D-Channel

“…Previous data-fitting protocols estimated the magnitude of the voltage generated during the slow phase to be about 3-fold larger than in the intermediate phase (17, 44, 45, 59). However, fitting of the data using a model of two consecutive, rather than two parallel protonic, electrogenic steps, shows that the intermediate and slow electrogenic phases have similar magnitudes (64, 66, 76). Hence, the intermediate and slow phases are likely to represent essentially the same proton transfer processes, namely, the two consecutive reprotonations of E286 via the D-channel (64, 76), with minor contributions from other coupled intraprotein charge movements.…”

“…However, fitting of the data using a model of two consecutive, rather than two parallel protonic, electrogenic steps, shows that the intermediate and slow electrogenic phases have similar magnitudes (64, 66, 76). Hence, the intermediate and slow phases are likely to represent essentially the same proton transfer processes, namely, the two consecutive reprotonations of E286 via the D-channel (64, 76), with minor contributions from other coupled intraprotein charge movements. In each of the “P M ”→F and F→O transitions, the first reprotonation occurs after the initially protonated E286-COOH donates its proton to the Pump Loading Site (protonic phase I).…”

Photosystem II and terminal respiratory oxidases molecular machines operating in opposite directions

“…But if the two protonic phases occur in series, rather than in parallel (which is likely to be the case), the true magnitude values need to be recalculated, according to refs. (28,274). The recalculated amplitudes of the two consecutive protonic phases are similar in magnitudes (A 2 /A 3 ~ 1:1.25) for both of the single-electron steps P M →F and F→O in the mitochondrial oxidase.…”

A quantum chemical study of the mechanism for proton-coupled electron transfer leading to proton pumping in cytochrome c oxidase

“…In some of those studies certain aspects of the pumping mechanism have been addressed. However, that type of issues have mainly been studied with simpler molecular mechanics and electrostatic methods [16][17][18][19][20][21][22][23]. Until very recently it was considered impossible to construct quantum chemical models that could give more direct information on the energetics of the proton pumping in terms of calculating reaction barriers involved in the gating mechanism.…”