Could a Diiron‐Containing Four‐Helix‐Bundle Protein Have Been a Primitive Oxygen Reductase?

Dioxygen, which became abundant in the earth's atmosphere approximately 2 ± 3 billion years ago, is a molecule with a very high oxidising power. Since dioxygen has a poor reactivityÐdue to unfavourable thermodynamic and kinetic factorsÐliving organisms could only take advantage of the oxic atmosphere by developing appropriate enzymes that can efficiently utilise it, that is, developing safe high-energy-yielding processes. At the present time, a large variety of proteins that are capable of utilising either dioxygen or its reduced species is known, and their evolution, as well as that of the metabolic pathways they integrate, is a challenging issue. The present work raises the hypothesis that a four-helix-bundle diiron-containing protein may have been an early oxygen reductase operating in the first stages of the transition between the anoxic and the present oxic atmosphere. This protein would have the required features to perform dioxygen chemistry, namely the potential to harbour a binuclear transition metal site. Such a system allows the complete reduction of dioxygen to water and would play an important role in oxygen defence mechanisms in primordial anaerobes. This hypothesis is based on data here reported concerning the oxygen reductase activity of the diiron protein rubrerythrin, together with sequenceand structure-based data of protein phylogeny between this and other diiron proteins. Interestingly, analysis of the available data depicts an evolutionary relationship between such an early system and the alternative oxidases present in extant eukaryotes. Our knowledge about the diiron carboxylate protein family has expanded considerably in the last years, with an increase in both sequence and structure data. 4] This family comprises the R2 subunit of ribonucleotide reductase (RNR R2), the hydroxylase subunit of the soluble methane monooxygenase (MMOH), (bacterio)ferritin ((B)FR), rubrerythrin (RR), stearoyl-acyl carrier D desaturase (D desaturase) and hemerythrin (HR). In all cases, carboxylates and oxide or hydroxide ions bridge the diiron site, surrounded by a four-helix-bundle protein fold. These enzymes have quite a large functional diversity, but they have an oxygenactivating step in common that, with the exception of hemerythrin, drives subsequent redox reactions. Plant alternative oxidase (AOX) is a non-energy-transducing terminal oxygen reductase operating in mitochondria and chloroplasts, which is reduced by ubiquinol and catalyses the four-electron reduction of dioxygen to water. 6] Despite numerous efforts, a pure preparation of this protein could never be obtained, thus preventing the elucidation of the chemical properties of its catalytic site. On the basis of sequence comparisons of several alternative oxidases, it has been suggested that they contain a diiron site as a reactive centre in a four-helix-bundle conformation. This hypothesis was recently further analysed by Andersson and Nordlund who revised the initially proposed structural model. These authors, considering additional sequences from alternative oxidases, presented a new model for this protein which likens it even more to the diiron carboxylate proteins. The amino acid sequence identity between the diiron carboxylate proteins is in general too low to allow the use of a conventional phylogenetic analysis. However, structure-oriented local sequence alignments, comprising only the regions of the four helices aligned according to homologous residues in the metal-binding site, can successfully be used to infer general phylogenetic relationships between these proteins. This approach was followed and the study of more than 50 sequences from diiron carboxylate proteins was undertaken, combining sequence and structure data for the structurally conserved region of the four-helix bundle (Figure 1). These structure-