EPR spectroscopy of putative enzyme intermediates in the NO reductase and the auto-nitrosylation reaction of Desulfovibrio vulgaris hybrid cluster protein.

The hybrid cluster protein (Hcp) contains a unique 4Fe cluster that is a hybrid of μ-S and μ-O bridges. Escherichia coli Hcp has recently been found to carry NO reductase activity as well as S-nitrosylation activity in NO-based signaling. In other species, the physiological activity has not been established. No reaction mechanism of any Hcp has been proposed. Here, we show that Desulfovibrio vulgaris (Hildenborough) Hcp has nitric oxide reductase activity with benzyl viologen as electron donor. With EPR spectroscopy, we identify three unexpected putative reaction intermediates: both in reduced and oxidized Hcp, dinitrosyl iron complexes are formed. Also, the hybrid cluster in reduced Hcp, but not in oxidized Hcp, binds the product N O. Possible implications for a reaction mechanism are discussed.