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

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Gorbikova, E.; Samsonov, S.A.; Kalendar, R. Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy. Molecules 2020, 25, 3393.

Title: Probing the Proton-Loading Site of Cytochrome C Oxidase Using Time-Resolved Fourier Transform Infrared Spectroscopy
Author: Gorbikova, Elena; Samsonov, Sergey A.; Kalendar, Ruslan
Publisher: Multidisciplinary Digital Publishing Institute
Date: 2020-07-27
URI: http://hdl.handle.net/10138/318207
Abstract: Crystal structure analyses at atomic resolution and FTIR spectroscopic studies of cytochrome <i>c</i> 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 <i>c</i> 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 C<i>c</i>O enzymes with oxygen was monitored and analyzed. Seven infrared bands in the &ldquo;fast&rdquo; kinetic spectra were found based on the following three requirements: (1) they are present in the &ldquo;fast&rdquo; phases of N131V and D124N mutants, (2) they have reciprocal counterparts in the &ldquo;slow&rdquo; kinetic spectra in these mutants, and (3) they are absent in &ldquo;fast&rdquo; 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).


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