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Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

ABSTRACT: Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable O_H state, and a reductive phase, in which O_H is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if O_H is allowed to relax to the resting oxidized state (O), a redox equivalent to O_H, its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a₃ iron and Cu_B in the active site of the O state, like those in the O_H state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from O_H, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide insights into the proton translocation mechanism of CcO.

SUBMITTER: Ishigami I

PROVIDER: S-EPMC10505203 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

Ishigami Izumi I Sierra Raymond G RG Su Zhen Z Peck Ariana A Wang Cong C Poitevin Frederic F Lisova Stella S Hayes Brandon B Moss Frank R FR Boutet Sébastien S Sublett Robert E RE Yoon Chun Hong CH Yeh Syun-Ru SR Rousseau Denis L DL

Nature communications 20230916 1

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable O<sub>H</sub> state, and a reductive phase, in which O<sub>H</sub> is reduced back to the R state. During each phase, two ...[more]

PMID: 37717031

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Project description:Geobacter sulfurreducens possesses over 100 cytochromes that assure an effective electron transfer to the cell exterior. The most abundant group of cytochromes in this microorganism is the PpcA family, composed of five periplasmic triheme cytochromes with high structural homology and identical heme coordination (His-His). GSU0105 is a periplasmic triheme cytochrome synthetized by G. sulfurreducens in Fe(III)-reducing conditions but is not present in cultures grown on fumarate. This cytochrome has a low sequence identity with the PpcA family cytochromes and a different heme coordination, based on the analysis of its amino acid sequence. In this work, amino acid sequence analysis, site-directed mutagenesis, and complementary biophysical techniques, including ultraviolet-visible, circular dichroism, electron paramagnetic resonance, and nuclear magnetic resonance spectroscopies, were used to characterize GSU0105. The cytochrome has a low percentage of secondary structural elements, with features of α-helices and β-sheets. Nuclear magnetic resonance shows that the protein contains three low-spin hemes (Fe(II), S = 0) in the reduced state. Electron paramagnetic resonance shows that, in the oxidized state, one of the hemes becomes high-spin (Fe(III), S = 5/2), whereas the two others remain low-spin (Fe(III), S = 1/2). The data obtained also indicate that the heme groups have distinct axial coordination. The apparent midpoint reduction potential of GSU0105 (-154 mV) is pH independent in the physiological range. However, the pH modulates the reduction potential of the heme that undergoes the low- to high-spin interconversion. The reduction potential values of cytochrome GSU0105 are more distinct compared to those of the PpcA family members, providing the protein with a larger functional working redox potential range. Overall, the results obtained, together with an amino acid sequence analysis of different multiheme cytochrome families, indicate that GSU0105 is a member of a new group of triheme cytochromes.

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Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

Publications

Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

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