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

Abstract

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 N2O. Possible implications for a reaction mechanism are discussed.

Anaerobic bacterial response to nitric oxide stress: Widespread misconceptions and physiologically relevant responses

“…The paper by Hagen (2019) provided the first insight into a possible mechanism for the two contrasting activities of Hcp. Bulot et al (2019) showed that during anaerobic growth in the presence of nitrate, both the nitrate reductase, NarGHI, and its cognate formate dehydrogenase, FdnGHI, cluster at the poles of the bacteria, consistent with the formation of a multi-protein complex.…”

“…Throughout three decades of research that produced high‐resolution structural and spectroscopic information, the physiological role of Hcp remained unknown. Four roles have been proposed, two of which can immediately be discarded (Hagen, 2019). A catalytically ineffective hydroxylamine reductase activity was demonstrated for the E. coli Hcp (Wolfe et al., 2002).…”

“…Despite warnings by the authors that this was unlikely to be its physiological function, Hcp is still annotated in many genomes as a hydroxylamine reductase. For similar reasons, a proposal that it provides defense against peroxide stress can be discounted (Almeida et al, 2006; Hagen, 2019).…”

“…(2016) has correctly been challenged. Hagen (2019) presented spectroscopic evidence that the D. vulgaris Hcp is indeed an NO reductase, but the key intermediate is a dinitrosyl Fe intermediate. This provides a potential mechanism to explain how Hcp can bind and reduce 2 NO molecules to generate N 2 O.…”

“…It also provides a possible mechanistic explanation for protein −SH nitrosation catalyzed by Hcp. By analogy with other iron‐sulfur proteins, Hagen (2019) proposed that the hybrid center of Hcp can exist in four redox states that he designated reduced, semi‐reduced, oxidized (the form in which Hcp was isolated) and super‐oxidized. Two molecules of NO can bind sequentially to either the oxidized or reduced forms of Hcp, though little, if any, mononitrosyl Hcp was detected spectroscopically even with a 2:1 ratio of Hcp to NO.…”