Filling the Catalytic Site of Cytochrome c Oxidase with Electrons

Abstract

In the reductive phase of its catalytic cycle, cytochrome c oxidase receives electrons from external electron donors. Two electrons have to be transferred into the catalytic center, composed of heme a 3 and Cu B , before reaction with oxygen takes place. In addition, this phase of catalysis appears to be involved in proton translocation. Here, we report for the first time the kinetics of electron transfer to both heme a 3 and Cu B during the transition from the oxidized to the fully reduced state. The state of reduction of both heme a 3 and Cu B was monitored by a combination of EPR spectroscopy, the rapid freeze procedure, and the stoppedflow method. The kinetics of cytochrome c oxidase reduction by hexaamineruthenium under anaerobic conditions revealed that the rate-limiting step is the initial electron transfer to the catalytic site that proceeds with apparently identical rates to both heme a 3 and Cu B . After Cu B is reduced, electron transfer to oxidized heme a 3 is enhanced relative to the rate of entry of the first electron.Mitochondrial cytochrome c oxidase (CcO) 4 is a membrane protein that catalyzes the oxidation of ferrocytochrome c by molecular oxygen. The reduction of oxygen to water requires the delivery of four electrons and four protons into the catalytic center of the enzyme. Electrons enter oxidase from the cytosolic domain and protons from the matrix side of the inner mitochondrial membrane. This redox reaction is coupled to the pumping of four additional protons across the membrane. Both of these processes contribute to the generation of a transmembrane proton gradient.Bovine heart CcO contains 13 polypeptides; however, four redox centers involved in electron transport (ET) and in the reduction of O 2 to water are located in two subunits (1). Three of these centers, heme a, heme a 3 , and copper ion called Cu B , are in subunit I, and the dinuclear copper center Cu A is located in subunit II (1).Cu A is close to the cytosolic surface of the protein and serves as the acceptor of electrons either from the physiological reductant, ferrocytochrome c, or artificial electron donors (2-6). Electrons received by the oxidase are rapidly distributed between Cu A and heme a on the microsecond time scale (3, 4, 6 -9). ET continues further to the catalytic center composed of heme a 3 and Cu B . At this catalytic center, the interaction of electrons, protons, and oxygen occurs. The reaction of CcO with oxygen, however, can take place only when two electrons have reduced the binuclear center. This part of the catalytic cycle of CcO, during which electrons are delivered to CcO prior to reaction with oxygen, is usually referred to as the reductive phase. It appears that the reductive phase consists not only of ET reactions but is accompanied also by proton pumping (10). Despite the significance of these ET reactions, there is virtually no data on how electrons are delivered into the catalytic site and how this ET is affected by the presumed redox interaction between heme a 3 and Cu B .Under anaerobic condi...

Reaction Mechanism of Cytochrome c Oxidase

“…The extremely weak sensitivity of the CO-stretch band of the bound CO on overall reduction potential also suggests the absence of a [Cu B 2+ , Fe a 3 2+ –CO] state, because Cu B 2+ /Cu B 1+ exchange would strongly influence the CO-stretch frequency . By analogy to the CO-binding, the complete reduction of the O 2 -reduction site is very likely to be prerequisite for O 2 binding. The infrared spectrum and X-ray structure of CO at Cu B indicate a fairly weak (essentially side-on) binding without any significant interaction with Fe a 3 . , However, the small but significant spectral changes in the 1 μs phase described above indicate that some significant interactions exist between Cu B and Fe a 3 , even when both metals are in the unliganded reduced state. A significant enhancement of electron transfer to Fe a 3 by anaerobic reduction of Cu B was revealed by stopped-flow freeze-quench EPR spectroscopic analyses The influence of the oxidation state of Cu B to the oxidized heme a 3 absorption spectrum has been confirmed by an extensive resonance Raman analyses …”

Synthetic Fe/Cu Complexes: Toward Understanding Heme-Copper Oxidase Structure and Function

“…The F and O H states as well as the F → O H transition have been a recent focus for extensive biophysical and computational analysis in which the role of water molecules and CuB redox potential are hotly debated, as comprehensive spectroscopic/structural data has not yet elucidated mechanistic details. 859,875,901–904 Two additional unresolved proton-coupled electron-transfers regenerate the TyrOH (forming the E H state, which has only been observed in electron injection experiments) 905 and finally the reduced state of the enzyme once again, completing the catalytic cycle and releasing the two water molecules as well as pumping two additional protons (Scheme 23). 906–908…”

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

“… 19 Another report by Fabian and co-workers provided evidence that the rate-limiting step is the initial electron transfer to the catalytic site. 20 The present results using biosynthetic models strongly suggest that the rate of ET into the active site plays a critical role in increasing the enzymatic activity in a mechanism like that of a native oxidase. A related and exciting area of research is to immobilize native enzymes and their variants onto electrodes to understand the protein–protein and protein–electrode interactions for efficient ET in applications such as biofuel cells.…”