Functional design of bacterial superoxide:quinone oxidoreductase

The superoxide anion - molecular oxygen reduced by a single electron - is produced in large amounts by enzymatic and adventitious reactions and can perform a range of cellular functions, including bacterial warfare and iron uptake, signalling and host immune response in eukaryotes. However, it also serves as precursor for more deleterious species such as the hydroxyl anion or peroxynitrite and therefore, cellular defense mechanisms for superoxide neutralization have evolved. In addition to the soluble proteins superoxide dismutase and superoxide reductase, recently the membrane embedded diheme cytochrome b561 (CybB) from E. coli has been proposed to act as a superoxide:quinone oxidoreductase. Here, we confirm superoxide and cellular ubiquinones or menaquinones as natural substrates and show that quinone binding to the enzyme accelerates the reaction with superoxide. The reactivity of the substrates is in accordance with the here determined midpoint potential of the two b hemes (+48 and -23 mV / NHE). Our data suggest that the enzyme can work near the diffusion limit in the forward direction and can also catalyse the reverse reaction efficiently under physiological conditions. The data is discussed in context of described cytochrome b561 proteins and potential physiological roles of CybB.

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

Evolution of b-type cytochromes in prokaryotes

Prokaryotes use a wide variety of bioenergetic pathways but the order of emergence of these pathways and their evolutionary relationships are still unresolved issues. In this study we focus on the evolutionary relationships of different families of b-type cytochromes, which form part of a variety of bioenergetic enzymes (the cytochrome b6f complex, ubiquinol and menaquinol reductases, formate dehydrogenase, Ni/Fe-hydrogenase, and succinate dehydrogenase). We use data from 272 species of fully sequenced bacteria and archaea, which represent the full diversity of prokaryotic lineages and multiple bioenergetic modes, to examine the distribution of these cytochromes across lineages, and ask the question of whether sequences from different species cluster by cytochrome-b family, by bioenergetic mode or by taxonomic group. Different cytochrome-b types are found in many lineages of the bacteria and archaea, and form distinct groups in phylogenetic analysis, which indicates an ancient origin for this complex, and diversification of different cytochrome-b types before the diversification of lineages. We find that species do not cluster based on bioenergetic mode. We also re-examine data from previous studies using this expanded sample of organisms spanning the full diversity of prokaryotic lineages. Concerning the b6f complex of photosynthetic organisms, our expanded phylogenies do not show significant bootstrap support for a "green clade" of cytochrome b6; also, the split form of b6 is not monophyletic, indicating that the split form arose independently multiple times. We also present data on the similarities between prokaryotic and eukaryotic cytochrome b561 sequences, in light of the recently reported structure of eukaryotic cytochrome b561.

Evolution of b-type cytochromes in prokaryotes

Prokaryotes use a wide variety of bioenergetic pathways but the order of emergence of these pathways and their evolutionary relationships are still unresolved issues. In this study we focus on the evolutionary relationships of different families of b-type cytochromes, which form part of a variety of bioenergetic enzymes (the cytochrome b6f complex, ubiquinol and menaquinol reductases, formate dehydrogenase, Ni/Fe-hydrogenase, and succinate dehydrogenase). We use data from 272 species of fully sequenced bacteria and archaea, which represent the full diversity of prokaryotic lineages and multiple bioenergetic modes, to examine the distribution of these cytochromes across lineages, and ask the question of whether sequences from different species cluster by cytochrome-b family, by bioenergetic mode or by taxonomic group. Different cytochrome-b types are found in many lineages of the bacteria and archaea, and form distinct groups in phylogenetic analysis, which indicates an ancient origin for this complex, and diversification of different cytochrome-b types before the diversification of lineages. We find that species do not cluster based on bioenergetic mode. We also re-examine data from previous studies using this expanded sample of organisms spanning the full diversity of prokaryotic lineages. Concerning the b6f complex of photosynthetic organisms, our expanded phylogenies do not show significant bootstrap support for a "green clade" of cytochrome b6; also, the split form of b6 is not monophyletic, indicating that the split form arose independently multiple times. We also present data on the similarities between prokaryotic and eukaryotic cytochrome b561 sequences, in light of the recently reported structure of eukaryotic cytochrome b561.