Reversible temperature-dependent high- to low-spin transition in the heme Fe–Cu binuclear center of cytochrome ba3 oxidase

A reversible temperature-dependent high- to low-spin transition with T1/2 = −60 °C has been observed in the resonance Raman spectra of the equilibrium reduced and photoreduced heme a3 of the thermophilic ba3 heme–copper oxidoreductase.

Comparison between O and OH intermediates of cytochrome c oxidase studied by FTIR spectroscopy

Cytochrome c oxidase is terminal enzyme in the respiratory chain of mitochondria and many aerobic bacteria. It catalyzes reduction of oxygen to water. During its catalysis, CcO proceeds through several quite stable intermediates (R, A, PR/M, O/OH, E/EH). This work is concentrated on the elucidation of the differences between structures of oxidized intermediates O and OH in different CcO variants and at different pH values. Oxidized intermediates of wild type and mutated CcO from Paracoccus denitrificans were studied by means of static and time-resolved Fourier-transform infrared spectroscopy in acidic and alkaline conditions in the infrared region 1800-1000 cm-1. No reasonable differences were found between all variants in these conditions, and in this spectral region. This finding means that the binuclear center of oxygen reduction keeps a very similar structure and holds the same ligands in the studied conditions. The further investigation in search of differences should be performed in the 4000-2000 cm-1 IR region where water ligands absorb.AbbreviationsAferrous-oxy intermediateATRAttenuated total reflectanceBGbackgroundBNCbinuclear centerCcOCytochrome c oxidaseEone-electron reduced intermediateFferryl intermediateFRCOfully-reduced CO-inhibited enzymeFTIRFourier transform infraredIRinfraredNHENormal Hydrogen ElectrodeN-sidenegatively charged side of the membranePR/M“peroxy”intermediates; P-sidepositively charged side of the membraneRfully-reduced intermediateTRtime-resolvedWTwild type

Multiscale simulations reveal key features of the proton-pumping mechanism in cytochrome c oxidase

Cytochrome c oxidase (CcO) reduces oxygen to water and uses the released free energy to pump protons across the membrane. We have used multiscale reactive molecular dynamics simulations to explicitly characterize (with free-energy profiles and calculated rates) the internal proton transport events that enable proton pumping during first steps of oxidation of the fully reduced enzyme. Our results show that proton transport from amino acid residue E286 to both the pump loading site (PLS) and to the binuclear center (BNC) are thermodynamically driven by electron transfer from heme a to the BNC, but that the former (i.e., pumping) is kinetically favored whereas the latter (i.e., transfer of the chemical proton) is rate-limiting. The calculated rates agree with experimental measurements. The backflow of the pumped proton from the PLS to E286 and from E286 to the inside of the membrane is prevented by large free-energy barriers for the backflow reactions. Proton transport from E286 to the PLS through the hydrophobic cavity and from D132 to E286 through the D-channel are found to be strongly coupled to dynamical hydration changes in the corresponding pathways and, importantly, vice versa.