The causes of reduced proton-pumping efficiency in type B and C respiratory heme-copper oxidases, and in some mutated variants of type A.

The heme-copper oxidases may be divided into three categories, A, B, and C, which include cytochrome c and quinol-oxidising enzymes. All three types are known to be proton pumps and are found in prokaryotes, whereas eukaryotes only contain A-type cytochrome c oxidase in their inner mitochondrial membrane. However, the bacterial B- and C-type enzymes have often been reported to pump protons with an H(+)/e(-) ratio of only one half of the unit stoichiometry in the A-type enzyme. We will show here that these observations are likely to be the result of difficulties with the measuring technique together with a higher sensitivity of the B- and C-type enzymes to the protonmotive force that opposes pumping. We find that under optimal conditions the H(+)/e(-) ratio is close to unity in all the three heme-copper oxidase subfamilies. A higher tendency for proton leak in the B- and C-type enzymes may result from less efficient gating of a proton pump mechanism that we suggest evolved before the so-called D-channel of proton transfer. There is also a discrepancy between results using whole bacterial cells vs. phospholipid vesicles inlaid with oxidase with respect to the observed proton pumping after modification of the D-channel residue asparagine-139 (Rhodobacter sphaeroides numbering) to aspartate in A-type cytochrome c oxidase. This discrepancy might also be explained by a higher sensitivity of proton pumping to protonmotive force in the mutated variant. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Mechanistic stoichiometry of proton translocation by cytochrome cbb3.

Cytochrome cbb(3) belongs to the superfamily of respiratory heme-copper oxidases that couple the reduction of molecular oxygen to proton translocation across the bacterial or mitochondrial membrane. The cbb(3)-type enzymes are found only in bacteria, and are both structurally and functionally the most distant from their mitochondrial counterparts. The mechanistic H(+)/e(-) stoichiometry of proton translocation in these cbb(3)-type cytochrome c oxidases has remained controversial. A stoichiometric efficiency of only one-half that of the mitochondrial aa(3)-type enzyme was recently proposed to be related to adaptation of the organism to microaerobic environments. Here, proton translocation by the Rhodobacter sphaeroides enzyme was studied using purified cytochrome cbb(3) reconstituted into liposomes. An H(+)/e(-) stoichiometry of proton translocation close to unity was observed using the oxygen pulse method, but solely in conditions in which the vast majority of the enzyme was fully reduced in the anaerobic state before the O(2) pulse. These data were compared with results using whole cells or spheroplasts, and the discrepancies in the literature data were discussed. Our results suggest that a proton-pumping efficiency of 1 H(+)/e(-) may be achieved using the single-proton uptake pathway identified in the structure of cytochrome cbb(3). The mechanism of proton pumping thus differs from that of the aa(3)-type oxidases of mitochondria and bacteria.

Initiation of the proton pump of cytochrome c oxidase.

Cytochrome c oxidase is the terminal enzyme of the respiratory chain that is responsible for biological energy conversion in mitochondria and aerobic bacteria. The membrane-bound enzyme converts free energy from oxygen reduction to an electrochemical proton gradient by functioning as a redox-coupled proton pump. Although the 3D structure and functional studies have revealed proton conducting pathways in the enzyme interior, the location of proton donor and acceptor groups are not fully identified. We show here by time-resolved optical and FTIR spectroscopy combined with time-resolved electrometry that some mutant enzymes incapable of proton pumping nevertheless initiate catalysis by proton transfer to a proton-loading site. A conserved tyrosine in the so-called D-channel is identified as a potential proton donor that determines the efficiency of this reaction.

Differences in the aerobic capacity of flight muscles between butterfly populations and species with dissimilar flight abilities.

Habitat loss and climate change are rapidly converting natural habitats and thereby increasing the significance of dispersal capacity for vulnerable species. Flight is necessary for dispersal in many insects, and differences in dispersal capacity may reflect dissimilarities in flight muscle aerobic capacity. In a large metapopulation of the Glanville fritillary butterfly in the Åland Islands in Finland, adults disperse frequently between small local populations. Individuals found in newly established populations have higher flight metabolic rates and field-measured dispersal distances than butterflies in old populations. To assess possible differences in flight muscle aerobic capacity among Glanville fritillary populations, enzyme activities and tissue concentrations of the mitochondrial protein Cytochrome-c Oxidase (CytOx) were measured and compared with four other species of Nymphalid butterflies. Flight muscle structure and mitochondrial density were also examined in the Glanville fritillary and a long-distance migrant, the red admiral. Glanville fritillaries from new populations had significantly higher aerobic capacities than individuals from old populations. Comparing the different species, strong-flying butterfly species had higher flight muscle CytOx content and enzymatic activity than short-distance fliers, and mitochondria were larger and more numerous in the flight muscle of the red admiral than the Glanville fritillary. These results suggest that superior dispersal capacity of butterflies in new populations of the Glanville fritillary is due in part to greater aerobic capacity, though this species has a low aerobic capacity in general when compared with known strong fliers. Low aerobic capacity may limit dispersal ability of the Glanville fritillary.

Active site of cytochrome cbb3.

Cytochrome cbb(3) is the most distant member of the heme-copper oxidase family still retaining the following major feature typical of these enzymes: reduction of molecular oxygen to water coupled to proton translocation across the membrane. The thermodynamic properties of the six redox centers, five hemes and a copper ion, in cytochrome cbb(3) from Rhodobacter sphaeroides were studied using optical and EPR spectroscopy. The low spin heme b in the catalytic subunit was shown to have the highest midpoint redox potential (E(m)(,7) +418 mV), whereas the three hemes c in the two other subunits titrated with apparent midpoint redox potentials of +351, +320, and +234 mV. The active site high spin heme b(3) has a very low potential (E(m)(,7) -59 mV) as opposed to the copper center (Cu(B)), which has a high potential (E(m)(,7) +330 mV). The EPR spectrum of the ferric heme b(3) has rhombic symmetry. To explain the origins of the rhombicity, the Glu-383 residue located on the proximal side of heme b(3) was mutated to aspartate and to glutamine. The latter mutation caused a 10 nm blue shift in the optical reduced minus oxidized heme b(3) spectrum, and a dramatic change of the EPR signal toward more axial symmetry, whereas mutation to aspartate had far less severe consequences. These results strongly suggest that Glu-383 is involved in hydrogen bonding to the proximal His-405 ligand of heme b(3), a unique interaction among heme-copper oxidases.

Identification of a histidine-tyrosine cross-link in the active site of the cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides.

The heme-copper oxidases constitute a superfamily of terminal dioxygen-reducing enzymes located in the inner mitochondrial or in the bacterial cell membrane. The presence of a mechanistically important covalent bond between a histidine ligand of the copper ion (Cu(B)) in the active site and a generally conserved tyrosine residue nearby has been shown to exist in the canonical cytochrome c oxidases. However, according to sequence alignment studies, this critical tyrosine is missing from the subfamily of cbb(3)-type oxidases found in certain bacteria. Recently, homology modeling has suggested that a tyrosine residue located in a different helix might fulfill this role in these enzymes. Here, we show directly by methods of protein chemistry and mass spectrometry that there is indeed a covalent link between this tyrosine and the copper-ligating histidine. The identity of the cross-linked tyrosine was determined by showing that the cross-link is not formed when this residue is replaced by phenylalanine, even though structural integrity is maintained. These results suggest a universal functional importance of the histidine-tyrosine cross-link in the mechanism of O(2) reduction by all heme-copper oxidases.