Time-resolved generation of membrane potential by ba cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and OH states

Abstract

Two electrogenic phases with characteristic times of ~14μs and ~290μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized "relaxed" O state of ba oxidase from T. thermophilus (O→E transition). The rapid phase reflects electron redistribution between Cu and heme b. The slow phase includes electron redistribution from both Cu and heme b to heme a, and electrogenic proton transfer coupled to reduction of heme a. The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a is reduced, but there is no proton pumping and no reduction of Cu. Single-electron reduction of the oxidized "unrelaxed" state (O→E transition) is accompanied by electrogenic reduction of the heme b/heme a pair by Cu in a "fast" phase (~22μs) and transfer of protons in "middle" and "slow" electrogenic phases (~0.185ms and ~0.78ms) coupled to electron redistribution from the heme b/heme a pair to the Cu site. The "middle" and "slow" electrogenic phases seem to be associated with transfer of protons to the proton-loading site (PLS) of the proton pump, but when all injected electrons reach Cu the electronic charge appears to be compensated by back-leakage of the protons from the PLS into the binuclear site. Thus proton pumping occurs only to the extent of ~0.1 H/e, probably due to the formed membrane potential in the experiment.

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

“…As discussed above, Wikström and co-workers propose that, in the conversion of the O H to the E H state, a Cu II -OH species in the heterobinuclear active site possibly forms in the O H state for ba 3 -type C c O. 1184 However, upon reduction of Cu II to Cu I , the hydroxide ligand is transferred to the iron center, resulting in Fe III -OH···Cu I species. Because of this, as well as accounting for the considerable amount of computational evidence for the formation of an Fe III -(OH)-Cu II species, we suggest that reduction of O H to E H in C c O via electron transfer from heme a 3 to Cu B occurs through a bridging hydroxide ligand (Scheme 27) (i.e., the O H to E H conversion involves an “inner-sphere” electron-transfer mechanism).…”

“…Electron transfer from heme b to heme a 3 in turn results in reduction of Cu B via ET from heme a 3 , giving the E H state (Scheme 26B). 1184…”

“… a See the text and ref 1184 for details. Colors indicate oxidation state of the metals (orange for Cu 1 , green for Cu II , red for Fe II , brown for Fe III , and brown/red for partially reduced Fe sites).…”

Coupled transport of electrons and protons in a bacterial cytochromecoxidase—DFT calculated properties compared to structures and spectroscopies

“…As depicted in Fig. 6 O + 1e À -E. 94,95 In the following redox step which regenerates State R, one additional electron is added to the active site, and if needed, a proton may move into the active site vicinity to protonate an OH À . The kinetics seen for O H -E H is different from that observed for O -E. The first gives more efficient electron transfer, with the electron terminating at Cu B , and proton transfer is more effective as well for both ba 3 and aa 3 type enzymes.…”

Proton Pumping and Non-Pumping Terminal Respiratory Oxidases: Active Sites Intermediates of These Molecular Machines and Their Derivatives

“…Where cyt c is cytochrome c ; QH 2 and Q are two-electron-reduced and oxidized forms of quinone (ubiquinone or menaquinone), respectively [ 12 , 25 ]; n~2–4 is the amount of the pumped protons in the B family HCOs [ 26 , 27 ]; H + in and H + out are the protons taken up from the N phase and released to the P phase, correspondingly.…”