The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a 3 (Fe 4؉ ‫؍‬ O 2؊ ) to the oxidized form (Fe 3؉ OH ؊ ). This redox step requires the delivery of a ''chemical'' H ؉ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR’s extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.

The kinetics of the oxidation of fully-reduced ba(3) cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa(3)-type oxidases. However the F intermediate in ba(3) oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P-->F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F-->O transition in ba(3) oxidase is close to that observed during the F-->O transition in the aa(3) oxidases. However, the P(R)-->F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.

Cytochrome c oxidase (COX) 1 is the terminal enzyme of the aerobic respiratory chain in mitochondria and in many prokaryotes (1-4). The enzyme conducts electrons from cytochrome c to O 2 and couples the energetically downhill electron transfer to the uphill electrogenic movement of protons across the membrane from the bacterial cytoplasm (or mitochondrial matrix) to the periplasm (or mitochondrial intermembrane space).The transmembrane charge separation by COX involves two different mechanisms. Half of the free energy (corresponding to the transmembrane translocation of four elementary electric charges per dioxygen reduced) is conserved by a direct mechanism; the four electrons and four protons required to convert O 2 to water come from opposite sides of the membrane. This direct mechanism was postulated by Mitchell (5, 6) in the original chemiosmotic hypothesis. Charge separation via this mechanism is an intrinsic part of the reaction chemistry and cannot be dissociated from it without re-engineering the proton and/or electron delivery pathways to the active site. Any mutation resulting in inhibition of this electrogenic function would inevitably inhibit electron transfer activity of the enzyme.The other half of the free energy is conserved by virtue of "proton pumping" discovered by Wikström in 1977 (7) and resulting in the transmembrane electrogenic translocation of four more protons per catalytic cycle. This proton pumping occurs by an "indirect" coupling mechanism and is not an obligatory part of the reaction chemistry. In principle, residues constituting the proton pump may be located in a separate part of the protein from the catalytic center, and the coupling can occur by virtue of finely tuned mechano-electrical interactions mediated by minor conformational changes of the protein (cf. Refs. 8 and 9).The indirect coupling of the proton pump has been demonstrated recently by the isolation of cytochrome oxidase mutants that have full catalytic electron transfer activity but that do not pump protons. This was first shown for the N131D mutant of the COX from Paracoccus denitrificans (10), which places an aspartic acid residue near the entrance of the D-channel. This mutant COX has wild type oxidase activity but does not pump protons (10), which is exactly the phenotype expected for cytochrome oxidase in which electron transfer is decoupled from proton translocation at the molecular level. The equivalent mutant of the COX from Rhodobacter sphaeroides, N139D,