Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy.

Elena Gorbikova,Ruslan Kalendar,Sergey A Samsonov

doi:10.3390/molecules25153393

Elena Gorbikova, Ruslan Kalendar + Show 1 more

Open Access

https://doi.org/10.3390/molecules25153393

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Abstract

Crystal structure analyses at atomic resolution and FTIR spectroscopic studies of cytochrome c oxidase have yet not revealed protonation or deprotonation of key sites of proton transfer in a time-resolved mode. Here, a sensitive technique to detect protolytic transitions is employed. In this work, probing a proton-loading site of cytochrome c oxidase from Paracoccus denitrificans with time-resolved Fourier transform infrared spectroscopy is presented for the first time. For this purpose, variants with single-site mutations of N131V, D124N, and E278Q, the key residues in the D-channel, were studied. The reaction of mutated CcO enzymes with oxygen was monitored and analyzed. Seven infrared bands in the “fast” kinetic spectra were found based on the following three requirements: (1) they are present in the “fast” phases of N131V and D124N mutants, (2) they have reciprocal counterparts in the “slow” kinetic spectra in these mutants, and (3) they are absent in “fast” kinetic spectra of the E278Q mutant. Moreover, the double-difference spectra between the first two mutants and E278Q revealed more IR bands that may belong to the proton-loading site protolytic transitions. From these results, it is assumed that several polar residues and/or water molecule cluster(s) share a proton as a proton-loading site. This site can be propionate itself (holding only a fraction of H+), His403, and/or water cluster(s).

Highlights

Cytochrome c oxidase (CcO) is the terminal complex (Complex IV) of the respiratory chain of mitochondria, many aerobic bacteria, and archaea
The double-difference spectra between the first two mutants and E278Q revealed more IR bands that may belong to the proton-loading site protolytic transitions
One clear IR band is present in the “fast” kinetic spectra of N131V at both pH values and D124N

Summary

Introduction

Cytochrome c oxidase (CcO) is the terminal complex (Complex IV) of the respiratory chain of mitochondria, many aerobic bacteria, and archaea. This reaction requires four electrons and four protons (“chemical” protons) to be sent to the BNC. CcO accepts electrons from a small soluble enzyme Cytochrome c from the outside of the membrane (P-side or positively charged side, see Figure 1) and protons from the matrix of mitochondria or cytosol of bacteria (N-side or negatively charged side, see Figure 1). This reaction is highly exergonic, and energy released is used to translocate four more protons (“pumped” protons) across the membrane (from the N-side to the P-side). CcO takes part in the production of a transmembrane electrochemical gradient (∆μH+ ) that is used for Adenosine Triphosphate (ATP)

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