Characterization of Two Dissimilatory Sulfite Reductases (Desulforubidin and Desulfoviridin) from the Sulfate-Reducing Bacteria. Mossbauer and EPR Studies

J. Legall, H. D. Peck, D. V. Dervartanian, I. Moura, A. R. Lino, A. V. Xavier, José J. G. Moura, G. Fauque, B. H. Huynh

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Abstract

Mössbauer, EPR, optical, and biochemical techniques were used to characterize the prosthetic groups of two dissimilatory sulfite reductases: desulforubidin from Desulfovibrio baculatus strain DSM 1743 and desulfoviridin from Desulfovibrio gigas. For each molecule of desulforubidin, which has an α2β2configuration, there are two sirohemes and four [4Fe-4S] clusters. The [4Fe-4S] clusters are in the diamagnetic 2+ oxidation state and exhibit Mossbauer spectral properties similar to those of the oxidized Bacillus stearothermophilus ferredoxin. The sirohemes are high-spin ferric (S = 5/2) and exhibit characteristic ferric heme EPR resonances at g = 6.43, 5.34, and 1.97. The Mössbauer parameters for the sirohemes (Δeq =1.94 ± 0.03 mm/s and δ = 0.42 ± 0.02 mm/s at 195 K) are consistent with a high-spin ferric heme assignment. The Mössbauer measurements further demonstrate that each siroheme is exchange-coupled to a [4Fe-4S]2+ cluster. Such an exchange-coupled siroheme-[4Fe-4S] unit has also been found in the assimilatory sulfite reductase from Escherichia coli (Christner, J. A., et al. J. Biol. Chem. 1981, 256, 2098–2101) and in a low molecular weight sulfite reductase from Desulfovibrio vulgaris (Huynh, B. H., et al. J. Biol. Chem. 1984, 259, 15373–15 376). Detailed data analysis suggests that even though the siroheme and the exchange-coupled [4Fe-4S] cluster in desulforubidin have spectral properties distinctively different from those of E. coli sulfite reductase, the exchange-coupling mechanism appears to be the same in both enzymes. Desulforubidin can be reduced under a hydrogen atmosphere in the presence of trace amounts of hydrogenase and methylviologen. The reducing electron was found to reside on the siroheme. The Mössbauer parameters for the reduced siroheme (Δeq =2.72 ± 0.05 mm/s and δ = 0.92 ± 0.03 mm/s at 4.2 K) indicate that it is in a high-spin ferrous (S = 2) state. The electronic states of the exchange-coupled and the uncoupled [4Fe-4S] clusters are unaltered under this reducing condition. The most exciting and curious results were obtained from the studies of desulfoviridin. We found that for each molecule of desulfoviridin there are two tetrahydroporphyrin groups and four [4Fe-4S]2+ clusters. Most surprisingly, about 80% of the tetrahydroporphyrin groups do not contain iron. With the assumption that each molecule can have up to two tetrapyrrolic groups, our finding suggests that 60-80% of the purified desulfoviridin molecules may contain only metal-free tetrahydroporphyrins while 40-20% of the molecules may contain one to two sirohemes. Interestingly, the sirohemes are also exchange-coupled to [4Fe-4S]2+ clusters. Implications for the existence of metal-free tetrahydroporphyrins in the purified enzymes are discussed. Spectroscopic properties for the iron-containing prosthetic groups in desulfoviridin are essentially the same as those reported for desulforubidin. In addition to the tetrapyrrolic groups and the [4Fe-4S] clusters, a solitary iron center was also found in both dissimilatory sulfite reductases. In the as-purified reductases, this solitary iron is high-spin ferric. In the reduced enzymes, it is high-spin ferrous. The Mössbauer parameters for the reduced iron (Δfq = 3.2 mm/s and δ = 1.25 mm/s at 4.2 K) are consistent with octahedrally coordinated Fe(II) compounds with oxygenous and/or nitrogenous ligands. Whether this iron is adventitiously bound to the protein or has any physiological role is presently unclear.

Original languageEnglish
Pages (from-to)1075-1082
Number of pages8
JournalJournal of the American Chemical Society
Volume110
Issue number4
DOIs
Publication statusPublished - 1 Jan 1988

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