Internal Electron Transfer in Cytochrome c Oxidase Is Coupled to the Protonation of a Group Close to the Bimetallic Site

Abstract

Absorbance changes following CO dissociation by flash photolysis from mixed-valence cytochrome oxidase have been followed in the Soret and alpha regions. Apart from CO dissociation and recombination, three kinetic phases with rate constants in the range 10(5)-10(3) s-1 at pH 7.5 can be resolved in both spectral regions. The slowest one of these phases, which had earlier only been observed in the alpha region, has now been detected in the Soret region by the use of a low CO concentration to slow down the recombination reaction. This phase had been assigned to a structural change, but a kinetic difference spectrum demonstrates that it represents electron transfer from cytochrome a3 to cytochrome a. A kinetic deuterium isotope effect of 2-3 at pH 7.5 suggests that it involves proton transfer as well. The temperature dependence of the reaction gives an Arrhenius activation energy of 42 kJ.mol-1. The reaction is faster at low pH, and the equilibrium is shifted toward cytochrome a as the pH is raised. The rate and equilibrium changes can be described as involving acid-base groups with pKa values of approximately 7.7 and 8.7, respectively. The kinetic results can be simulated on the basis of a model in which one acid-base group interacts with cytochrome a3, so that its pKa drops on oxidation of this center. The group is in proton equilibrium with the solvent via a proton pathway, suggested to be a proton channel. The rate of a shift in the redox equilibrium between the two cytochromes reaches a high limit at low pH, where the channel is saturated with protons.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

“…As found previously (15,18,19), the extent (absorption change amplitude) and rate of the ms-phase electron-transfer reaction were both strongly pH dependent. In addition, we have shown here that changes in conductance take place with the same time constant as the absorbance changes associated with electron transfer between cytochrome a3 and a.…”

“…These reactions are followed by a slower electron equilibration in which additional electrons are transferred from cytochrome a3 to a on a millisecond time scale (this reaction will be referred to as the ms phase), presumably limited by the proton release. As found both in the bovine (15,19) and Rhodobacter sphaeroides bacterial enzymes (18), both the extent and time constant of this reaction are pH dependent; the time constant increases from about 0.5 ms at pH 6 to about 6 ms at pH 10 and more electrons are transferred at high pH than at low pH (15,18,19). This behavior was modeled in terms of electrostatic interactions between the binuclear center and a protonatable group in the vicinity of cytochrome a3 (18,19), in contact with the bulk protons through a proton-conducting pathway, which limits the proton-exchange rate with the bulk and, as a consequence, also the rate of the ms-phase electron transfer from cytochrome a3 to a (19).…”

“…2A) with a pHdependent time constant and amplitude (Fig. 3), previously attributed to electron transfer from cytochrome a3 to a, due to interactions with a protonatable group (18,19). For example, at pH 8.4 the time constant was 2.2 ± 0.3 ms and at pH 10 the time constant was 6.2 ± 0.4 ms.…”

Nanosecond electron tunneling between the hemes in cytochrome bo 3

“…This reaction was originally reported to consist of two phases (5,6): a Ϸ3-s phase of eT between the heme groups was followed by slower electron equilibration with an additional copper site (Cu A ) that lies at the membrane interface in some of the heme-copper oxidases but that is lacking in others (7,8). More recently, it was shown that a third phase of interheme eT (50 s to milliseconds) that is coupled to proton release from the binuclear center can be observed after CO photolysis at high pH (9)(10)(11). However, with the quinol-oxidizing cytochrome bo 3 from Escherichia coli, even more phases were discerned (12).…”

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

“…10 and 11, there are two separate input channels in COX for the uptake of ''pumped'' and ''chemical'' protons. Evidence for electrogenic movement of protons within the channel involved in the protonation of the binuclear center from the mitochondrial matrix was reported (12,13) and ionization of groups within this channel is apparently coupled to electron transfer between hemes a and a 3 (14,15).…”