TY - JOUR
T1 - Probing the proton-loading site of cytochrome C oxidase using time-resolved fourier transform infrared spectroscopy
AU - Gorbikova, Elena
AU - Samsonov, Sergey A.
AU - Kalendar, Ruslan
N1 - Funding Information:
Funding: E.G. was supported by the Center of International Mobility (CIMO) and Informational and Structural Biology (ISB) Graduate School. The work was supported by the Sigrid Juselius foundation, Biocentrum Helsinki and the Academy of Finland (project numbers 200726, 44895, and 115108). S.A.S. was supported by the National Science Centre of Poland (UMO-2018/30/E/ST4/00037). Open access funding provided by University of Helsinki including Helsinki University Central Hospital. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/8
Y1 - 2020/8
N2 - 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).
AB - 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).
KW - Cytochrome c oxidase
KW - D-channel mutants
KW - FTIR spectroscopy
KW - Proton transfer
KW - Proton-loading site
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U2 - 10.3390/molecules25153393
DO - 10.3390/molecules25153393
M3 - Article
C2 - 32727022
AN - SCOPUS:85088851715
VL - 25
JO - Molecules
JF - Molecules
SN - 1420-3049
IS - 15
M1 - 3393
ER -