Simulating the slow to fast switch in cytochrome c oxidase catalysis by introducing a loop flip near the enzyme's cytochrome c (substrate) binding site

Abstract

The mitochondrial enzyme cytochrome c oxidase catalyzes the reduction of molecular oxygen in the critical step of oxidative phosphorylation that links the oxidation of food consumed to ATP production in cells. The enzyme catalyzes the reduction of oxygen at two vastly different rates that are thought to be linked to two different conformations but the conformation of the "fast enzyme" remains obscure. In this study, we demonstrated how oxygen binding at haem a3 could trigger long-distance conformational changes and then simulated a conformational change in an eight-residue loop near the enzyme's substrate (cytochrome c) binding site. We then used this modified cytochrome c oxidase (COX) to simulate a stable COX-cytochrome c enzyme-substrate (ES) complex. Compared to ES complexes formed in the absence of the conformation change, the distance between the redox centers of the two proteins was reduced by half and instead of nine, only four COX amino acid residues were found along the axis linking the electron entry point and the CuA redox center of COX: We proposed that intramolecular electron transfer in COX occurs via a charge/hydrogen relay system involving these four residues. We suggest that the conformational change and resulting shortened electron pathway are features of fast-acting COX.

Characterisation of the Cyanate Inhibited State of Cytochrome c Oxidase

“…Preparation of this state has therefore been done to study the structure of central intermediates in CcO 19 . Analysis of the binding kinetics of cyanide to the BNC furthermore led to the discovery of a "slow" and a "fast" form of the enzyme [20][21][22][23] . Hereby the "slow" form exhibits lower reactivity than the "fast" form for inhibitors 24 and, in addition, shows a slowed down intramolecular electron transfer 25 .…”

Complex Interplay of Heme-Copper Oxidases with Nitrite and Nitric Oxide

“…Inhibition of the BNC by CN − , H 2 S, and N 3 − has been intensively investigated, offering the best illustration of the mechanisms underpinning the oxygen-binding and proton pumping [98,99]. Three forms of mitochondrial aa 3 -HCOs in the O state have been defined by the difference in the cyanide binding rates, namely, "slow", "fast", and "open" forms [100,101]. During the catalytic turnover, the open form resulting from O 2 oxidation of the R state has Fe 3+ -OH − as the iron coordination structure of the O 2 reduction site, allowing proton pumping after each of the first and second single-electron donations to the fully oxidized enzyme [102].…”