Quantification of Local Electric Field Changes at the Active Site of Cytochrome c Oxidase by Fourier Transform Infrared Spectroelectrochemical Titrations

Abstract

Cytochrome c oxidase (CcO) is a transmembrane protein complex that reduces molecular oxygen to water while translocating protons across the mitochondrial membrane. Changes in the redox states of its cofactors trigger both O2 reduction and vectorial proton transfer, which includes a proton-loading site, yet unidentified. In this work, we exploited carbon monoxide (CO) as a vibrational Stark effect (VSE) probe at the binuclear center of CcO from Rhodobacter sphaeroides. The CO stretching frequency was monitored as a function of the electrical potential, using Fourier transform infrared (FTIR) absorption spectroelectrochemistry. We observed three different redox states (R4CO, R2CO, and O), determined their midpoint potential, and compared the resulting electric field to electrostatic calculations. A change in the local electric field strength of +2.9 MV/cm was derived, which was induced by the redox transition from R4CO to R2CO. We performed potential jump experiments to accumulate the R2CO and R4CO species and studied the FTIR difference spectra in the protein fingerprint region. The comparison of the experimental and computational results reveals that the key glutamic acid residue E286 is protonated in the observed states, and that its hydrogen-bonding environment is disturbed upon the redox transition of heme a3. Our experiments also suggest propionate A of heme a3 changing its protonation state in concert with the redox state of a second cofactor, heme a. This supports the role of propionic acid side chains as part of the proton-loading site.

Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy

“…We have previously reported a similar behavior of lipid ester bands in spectroelectrochemical titrations of Rs C c O embedded in hydrated DPPC layers ( Baserga et al, 2021 ). However, it was impossible to determine whether this contribution originated from protein activation or from the external electric field applied to the protein-lipid film ( Zawisza et al, 2007 ).…”

“…Cytochrome c oxidase from R. sphaeroides ( Rs C c O) is a model system for the aa 3 oxidase family of heme-copper enzymes ( García-Horsman et al, 1994 ; Pereira et al, 2001 ; Popovic et al, 2010 ). Some of the many observable changes in its cycle are a direct consequence of its redox reaction ( Heibel et al, 1993 ; Kozuch et al, 2013 ); some are due to the interaction of the protein and cofactors ( Sezer et al, 2017 ; Baserga et al, 2021 ); and some are related only to amino acid side-chains as derived by FTIR spectroscopy ( Hellwig et al, 1999 ; Heitbrink et al, 2002 ; Iwaki et al, 2002 ). It is likely that many of these changes also entail structural rearrangements ( Qin et al, 2009 ).…”

In Situ Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

“…Under steady state conditions, CO binds to the central iron ion of heme a 3 from where it may migrate to Cu B , that is, upon irradiation with visible light. − The IR bands of Fe–CO (1964 cm –1 ) and Cu–CO (2045 cm –1 ) are clearly different. Because of the redox cooperativity, oxidation of heme a in the MV state causes a 4 cm –1 upshift of the Fe–CO frequency at heme a 3 . This illustrates how CO ligands report on changes in redox and protonation state through space acting as Stark probes …”

“…In the “mixed valence” state (MV or R 2 ), Cu A and heme a are oxidized, which shifts the CO frequency at heme a 3 to higher energies. These shifts can be used to benchmark calculations of local electric field changes at the BNC . Cyanide primarily binds to the oxidized heme a 3 site (Fe 3+ ) and Cu B irrespective of redox state …”

“…The FR-CO state converts into the O-state. The “O 2 –CO” difference spectrum in the main panel shows a characteristic difference signature with the positive band at 1745 cm –1 hinting at protonation of Glu286 in the O-state . Although some of the remaining bands have been assigned, − ,− a comprehensive understanding of all observed differences, including hydrogen-bonding and protonation changes as well as protein structural and heme-associated changes is yet to achieve.…”