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.
Highlights
The eukaryotic respiratory chain exploits electron-rich substrates to pump protons from the mitochondrial matrix into the intermembrane space; the resulting proton gradient provides energy for adenosine triphosphate (ATP) production (Mitchell, 1966)
The blueshift of the carbon monoxide (CO) peak is characteristic of the formation of the R2CO state (Dodson et al, 1996; Iwaki and Rich, 2007), i.e., the state in which the binuclear center (BNC) is reduced, but the other metal centers in cytochrome c oxidase (CcO) are oxidized (Brzezinski and Malmström, 1985)
The identifiable states are: (I) a state where CO is initially bound under reducing conditions [R4CO with ν(C≡O) = 1963.7 cm−1], (II) a state in which the majority of the sample has undergone the CO band shift [R2CO with ν(C≡O) = 1967.2 cm−1], and (III) a state in which the CO ligand dissociates from the BNC (O) (Dodson et al, 1996; Cooper and Plotting the relative abundance of the two CO-inhibited species against the applied electrode potential illustrates the depletion and formation of the R4CO, R2CO, and O states (Figure 3C)
Summary
The eukaryotic respiratory chain exploits electron-rich substrates to pump protons from the mitochondrial matrix into the intermembrane space; the resulting proton gradient provides energy for adenosine triphosphate (ATP) production (Mitchell, 1966). The terminal oxidase from Rhodobacter sphaeroides (RsCcO) is often used as a model organism for eukaryotic isoenzymes, since its active site shares high-sequence identity to mammalian oxidases, e.g., from Bos taurus (BtCcO) (Hosler et al, 1992; García-Horsman et al, 1994). RsCcO comprises four subunits: subunit I is fundamental to the function of the enzyme as it harbors the two cofactors heme a and heme a3. The latter forms the catalytic binuclear center (BNC), together with a copper ion (CuB) that is coordinated by three histidine residues (Pereira et al, 2001)
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