Photoinduced Electron Transfer in the Cytochrome c/Cytochrome c Oxidase Complex Using Thiouredopyrenetrisulfonate-Labeled Cytochrome c. Optical Multichannel Detection

Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence Cu A center of subunit II. In Thermus thermophilus a soluble cytochrome c 552 acts as the electron donor to ba 3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c 552 . In the present study, soluble fragments of the Cu A domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c 552 and Cu A couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Brøn-sted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble Cu A domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases.

Section: Resultssupporting

confidence: 83%

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

Maneg

Ludwig

Malatesta

2003

“…Three of these centers, heme a, heme a 3 , and copper ion, called Cu B , are in subunit I and a dinuclear copper center Cu A , is located in subunit II (2). Cu A serves as the acceptor of electrons either from ferrocytochrome c or from artificial electron donors (3)(4)(5)(6)(7). Electrons received by the oxidase are rapidly distributed between Cu A and heme a on the microsecond time scale (4,5,(7)(8)(9)(10).…”

mentioning

confidence: 99%

“…Cu A serves as the acceptor of electrons either from ferrocytochrome c or from artificial electron donors (3)(4)(5)(6)(7). Electrons received by the oxidase are rapidly distributed between Cu A and heme a on the microsecond time scale (4,5,(7)(8)(9)(10). ET continues further to the catalytic center composed of heme a 3 and Cu B where O 2 is reduced to water.…”

mentioning

confidence: 99%

Spectral and Kinetic Equivalence of Oxidized Cytochrome c Oxidase as Isolated and “Activated” by Reoxidation

Jancura

Berka

Antalı́k

et al. 2006

The spectral and kinetic characteristics of two oxidized states of bovine heart cytochrome c oxidase (CcO) have been compared. The first is the oxidized state of enzyme isolated in the fast form (O) and the second is the form that is obtained immediately after oxidation of fully reduced CcO with O 2 (O H ). No observable differences were found between O and O H states in: (i) the rate of anaerobic reduction of heme a 3 for both the detergent-solubilized enzyme and for enzyme embedded in its natural membraneous environment, (ii) the one-electron distribution between heme a 3 and Cu B in the course of the full anaerobic reduction, (iii) the optical and (iv) EPR spectra. Within experimental error of these characteristics both forms are identical. Based on these observations it is concluded that the reduction potentials and the ligation states of heme a 3 and Cu B are the same for CcO in the O and O H states.Mitochondrial cytochrome c oxidase (CcO) 2 transfers electrons from cytochrome c to dioxygen. The reduction of O 2 to water is associated with the generation of a transmembrane proton gradient. Two processes contribute to the development of this gradient. The first is the oxidation of ferrocytochrome c from the cytosolic side of the inner mitochondrial membrane coupled to proton consumption for water formation from the matrix compartment of mitochondria. The second process is proton pumping during which protons are translocated from the matrix space to the cytosolic side of the membrane (1).In CcO four redox centers participate in electron transfer (ET) and reduction of O 2 to H 2 O. Three of these centers, heme a, heme a 3 , and copper ion, called Cu B , are in subunit I and a dinuclear copper center Cu A , is located in subunit II (2). Cu A serves as the acceptor of electrons either from ferrocytochrome c or from artificial electron donors (3-7). Electrons received by the oxidase are rapidly distributed between Cu A and heme a on the microsecond time scale (4, 5, 7-10). ET continues further to the catalytic center composed of heme a 3 and Cu B where O 2 is reduced to water.The part of the catalytic cycle during which electrons are delivered to CcO, prior to reaction with O 2 , is usually referred to as the reductive phase. In the subsequent oxidative phase the reduced enzyme reacts with O 2 . It was proposed (11) and demonstrated later that proton pumping takes place in both the reductive and oxidative phases (12)(13)(14). Two protons are pumped in the oxidative phase when fully reduced CcO is oxidized by oxygen (12,14). Subsequently, if this freshly oxidized enzyme is reduced by two electrons two additional protons are translocated across the membrane (12, 14).However, proton translocation in the reductive phase is only observed with freshly oxidized enzyme. This reoxidized enzyme appears to be in a metastable state, which relaxes relatively slowly in the absence of electron donors to the stable oxidized form on a time scale of seconds. These two forms of oxidized enzyme, CcO as it exists immediately after ox...

“…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.…”

mentioning

confidence: 99%

“…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)(3)(4)(5)(6). Electrons received by the oxidase are rapidly distributed between Cu A and heme a on the microsecond time scale (3, 4, 6 -9).…”

mentioning

confidence: 99%

Filling the Catalytic Site of Cytochrome c Oxidase with Electrons

Jancura

Antalı́k

Berka

et al. 2006

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...