Benet C. Prickril

IDENTIFICATION OF THREE CLASSES OF HYDROGENASE IN THE GENUS, DESULFOVIBRIO

A comparison of amino-terminal amino acid sequences from the large and small subunits of hydrogenases from Desulfovibrio reveals significant differences. These results, in conjunction with antibody analyses, clearly indicate that the iron, iron + nickel, and iron + nickel + selenium containing hydrogenases represent three distinct classes of hydrogenase in Desulfovibrio.

Intrapeptide sequence homology in rubrerythrin from Desulfovibrio vulgaris: identification of potential ligands to the diiron site.

Two regions in the amino acid sequence of the 21.5 kDa subunit of rubrerythrin from Desulfovibrio vulgaris (Hildenborough) are shown to be homologous. Rubrerythrin contains a non-heme, non-sulfur diiron site, and the internally homologous regions share homology with at least one proposed iron binding region of the component A alpha subunit of methane monooxygenase, which also contains a non-heme, non-sulfur diiron site. Comparison of the rubrerythrin sequences with those of the B2 subunit of E. coli ribonucleotide reductase, whose diiron site ligands have been identified, suggests that two glutamate and two histidine residues at positions 53, 56, 129, and 131 within the rubrerythrin sequence furnish ligands to the diiron site. A pair of EXXH sequences appears to represent a diiron binding motif in all three aforementioned proteins. No propene monooxygenase activity was detected with rubrerythrin using the assay designed to test activity of methane monooxygenase component A in the absence of other protein components.

Rubrerythrin: A non-heme iron protein with structural analogies to ribonucleotide reductase.

Rubrerythrin, a contraction of rubredoxin and hemerythrin, is the trivial name given to a non-heme iron protein isolated from Desulfovibrio vulgaris (Hildenborough). This protein, whose physiological function is unknown, was first characterized by J. LeGall et al. [(1988) Biochemistry 28, 1636] as being a homodimer of subunit M(r) = 21,900 with four Fe per homodimer distributed as two rubredoxin-type FeS4 centers and one hemerythrin-type diiron cluster. Subsequent analysis of the amino acid sequence of the rubrerythrin gene [Kurtz, D. M., Jr., & Prickril, B.C. (1991) Biochem. Biophys. Res. Commun. 181, 137] revealed an internal homology which suggested that each subunit can accommodate one diiron cluster. Here, we report a procedure for reconstitution of the as-isolated D. vulgaris rubrerythrin with 57Fe. The reconstituted protein was characterized by optical, electron paramagnetic resonance, and Mössbauer spectroscopies. The results indicate successful incorporation of 57Fe into the two types of sites and strongly suggest that each subunit of rubrerythrin can indeed accommodate one diiron cluster as well as one rubredoxin-type center. Combined with amino acid sequence analysis, the spectroscopic characterization further suggests that the rubrerythrin subunit contains a diiron site whose structure is more closely related to that in ribonucleotide reductase than to that in hemerythrin.