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ABSTRACT Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus , which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y ). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s −1 . Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s −1 . The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes. IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed. 

Dihydropyrimidine dehydrogenase (DPD) catalyzes the initial step in the catabolism of the pyrimidines uracil and thymine. Crystal structures have revealed an elaborate subunit architecture consisting of two flavin cofactors, apparently linked by four Fe4S4 centers. Analysis of the DPD reaction(s) equilibrium position under anaerobic conditions revealed a reaction that favors dihydropyrimidine formation. Single-turnover analysis shows biphasic kinetics. The serine variant of the candidate general acid, cysteine 671, provided enhanced kinetic resolution for these phases. In the first event, one subunit of the DPD dimer takes up two electrons from NADPH in a reductive activation step. Spectrophotometric deconvolution suggests that thes electrons reside on one of the two flavins. That oxidation of the enzyme by dioxygen can be suppressed by the addition of pyrimidine, is consistent with these electrons residing on the FMN. The second phase involves further oxidation of NADPH and concomitant reduction of the pyrimidine substrate. During this phase no net reduction of DPD cofactors is observed indicating that the entire cofactor set acts as a wire, transmitting electrons from NADPH to the pyrimidine rapidly. This indicates that the availability of the proton from C671 general acid controls the transmittance of electrons from NADPH to the pyrimidine. Acid quench and HPLC product analysis of single-turnover reactions with limiting NADPH confirmed 2:1, NADPH:pyrimidine stoichiometry for the enzyme accounting for successive activation and pyrimidine reduction. These data support an alternating subunit model in which one protomer is activated and turns over before the other subunit can be activated and enter catalysis.