Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase [Enzymology]

August 21st, 2015 by Wu, S.-Y., Rothery, R. A., Weiner, J. H.

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining Mo active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The Mo atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates which are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser719, NarG-His1163, and NarG-His1184); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His1092 and NarG-His1098). A Ser719Ala variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the His1163Ala and His1184Ala variants elicit large effects (deltaEm values of -88 mV and -36 mV, respectively). Ala variants of His1092 and His1098 also elicit large deltaEm values of -143 mV and -101 mV, respectively. An Arg variant of His1092 elicits a small deltaEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the Mo Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.