A cooperative model for protonmotive heme‐copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase

Bloch

Proc. Natl. Acad. Sci. U.S.A.

et al. 2007

214

294

Cytochrome c oxidase catalyzes most of the biological oxygen consumption on Earth, a process responsible for energy supply in aerobic organisms. This remarkable membrane-bound enzyme also converts free energy from O2 reduction to an electrochemical proton gradient by functioning as a redox-linked proton pump. Although the structures of several oxidases are known, the molecular mechanism of redox-linked proton translocation has remained elusive. Here, correlated internal electron and proton transfer reactions were tracked in real time by spectroscopic and electrometric techniques after laser-activated electron injection into the oxidized enzyme. The observed kinetics establish the long-sought reaction sequence of the proton pump mechanism and describe some of its thermodynamic properties. The 10-s electron transfer to heme a raises the pKa of a ''pump site,'' which is loaded by a proton from the inside of the membrane in 150 s. This loading increases the redox potentials of both hemes a and a3, which allows electron equilibration between them at the same rate. Then, in 0.8 ms, another proton is transferred from the inside to the heme a3/CuB center, and the electron is transferred to CuB. Finally, in 2.6 ms, the preloaded proton is released from the pump site to the opposite side of the membrane.cytochrome oxidase ͉ electron transfer ͉ proton translocation

Section: Assignment Of the Proton Transfer Reactionssupporting

confidence: 90%

Exploring the proton pump mechanism of cytochrome c oxidase in real time

Bloch

Proc. Natl. Acad. Sci. U.S.A.

et al. 2007

214

294

Proc. Natl. Acad. Sci. U.S.A.

“…Direct observation of proton transfer through the key structures of these pathways by time-resolved infrared spectroscopy is expected to provide the means of conclusively identifying the pathway. The possibility that the low spin heme functions as the driving element of the proton pumping process has been proposed on several occasions back to 1978 (33)(34)(35)(36).…”

Section: Resultsmentioning

confidence: 99%

The proton pumping pathway of bovine heart cytochrome c oxidase

Shimokata

Katayama

Murayama

et al. 2007

133

134

X-ray structures of bovine heart cytochrome c oxidase have suggested that the enzyme, which reduces O 2 in a process coupled with a proton pumping process, contains a proton pumping pathway (H-pathway) composed of a hydrogen bond network and a water channel located in tandem across the enzyme. The hydrogen bond network includes the peptide bond between Tyr-440 and Ser-441, which could facilitate unidirectional proton transfer. Replacement of a possible proton-ejecting aspartate (Asp-51) at one end of the H-pathway with asparagine, using a stable bovine gene expression system, abolishes the proton pumping activity without influencing the O 2 reduction function. Blockage of either the water channel by a double mutation (Val386Leu and Met390Trp) or proton transfer through the peptide by a Ser441Pro mutation was found to abolish the proton pumping activity without impairment of the O 2 reduction activity. These results significantly strengthen the proposal that H-pathway is involved in proton pumping. mutagenesis ͉ mitochondrial import ͉ HeLa cell ͉ peptide bond ͉ keto-enol tautomerism

Journal of Biological Chemistry

“…Papa et al (19) have suggested a regulatory function of this "direct" electron transfer reaction for the stoichiometry of proton pumping. They proposed that, even in the wild-type enzyme where, under normal conditions, Cu A -heme a electron transfer is at least 100-fold faster than Cu A -heme a 3 electron transfer, the latter may occur to a significant extent at high ⌬ H ϩ.…”

Section: Discussionmentioning

confidence: 99%

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Kannt¹,

Pfitzner²,

Ruitenberg³

et al. 1999

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a ␣-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a 3 . This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu A to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where ⌬ generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.