SUMMARY The importance of molybdoenzymes is exemplified both by the debilitating and fatal human diseases caused by their deficiency and by their persistence throughout evolution. Here, we show that the protein fold of the molybdopyranopterin-containing domain of sulfite oxidase (the SUOX fold) can be found in all three domains of life. Analyses of sequence data and protein structure comparisons (secondary structure matching) show that the SUOX fold is found in enzymes that have quite distinct macromolecular architectures comprising one or more domains and sometimes subsidiary subunits. These are summarized as follows: (i) animal SUOXs that contain an N-terminal cytochrome b 5 domain and an SUOX fold fused to a C-terminal dimerization domain; (ii) plant SUOX that contains an SUOX fold fused to a C-terminal dimerization domain; (iii) the YedY protein from Escherichia coli, which comprises only the SUOX fold; (iv) the sulfite dehydrogenase from Starkeya novella that contains the SUOX fold, a dimerization domain, and an additional c-type cytochrome subunit; and (v) the plant-type nitrate reductases, exemplified by that of Pichia angusta, that contain an N-terminal SUOX fold, a dimerization domain, a cytochrome b 5 domain, and a C-terminal NADH binding flavin adenine dinucleotide-containing domain. We used the primary sequences of the proteins containing an SUOX fold to mine 559 sequences of related proteins. A phylogeny of a nonredundant subset of these sequences was generated, and the resultant clades were categorized by sequence motif analyses in the context of the available protein structures. Based on the motif analyses, cladistics, and domain conservations, we are able to postulate a plausible pathway of SUOX fold enzyme evolution.

We report a structural characterization using X-ray absorption spectroscopy of the molybdenum site of Escherichia coli YedY, a novel oxidoreductase related to be the sulfite oxidase family of molybdenum enzymes. We find that the enzyme can exist in Mo(V) and Mo(IV) oxidation states but cannot be readily oxidized to the Mo(VI) form. Mo(V) YedY has molybdenum coordination similar to that of sulfite oxidase, with one Mo═O at 1.71 Å, three Mo-S at 2.39 Å, and one Mo-OH at 2.09 Å, which elongates to 2.20 Å upon reduction to Mo(IV), indicating Mo-OH(2) coordination. The Mo(V) enzyme also possesses a long Mo-O coordination at 2.64 Å, which may be due to oxygen coordination by Asn-45 O(δ), with Mo-O(δ) approximately trans to the Mo═O group. A comparison with sulfite oxidase indicates that YedY possesses a much more uniform Mo-S coordination, with a maximum permitted deviation of less than 0.05 Å. Our results indicate that the YedY active site shows considerable similarity to but also important differences from that of reduced forms of sulfite oxidase.