Abstract
A putative chromate ion binding site was identified proximal to a rigidly bound FMN from electron densities in the crystal structure of the quinone reductase from Gluconacetobacter hansenii (Gh-ChrR) (3s2y.pdb). To clarify the location of the chromate binding site, and to understand the role of FMN in the NADPH-dependent reduction of chromate, we have expressed and purified four mutant enzymes involving the site-specific substitution of individual side chains within the FMN binding pocket that form non-covalent bonds with the ribityl phosphate (i.e., S15A and R17A in loop 1 between β1 sheet and α1 helix) or the isoalloxanzine ring (E83A or Y84A in loop 4 between the β3 sheet and α4 helix). Mutations that selectively disrupt hydrogen bonds between either the N3 nitrogen on the isoalloxanzine ring (i.e., E83) or the ribitylphos- phoate (i.e., S15) respectively result in 50% or 70% reductions in catalytic rates of chromate reduction. In comparison, mutations that disrupt π-π ring stacking interactions with the isoal-loxanzine ring (i.e., Y84) or a salt bridge with the ribityl phosphate result in 87% and 97% inhibittion. In all cases there are minimal alterations in chromate binding affinities. Collectively, these results support the hypothesis that chromate binds proximal to FMN, and implicate a structural role for FMN positioning for optimal chromate reduction rates. As side chains proximal to the β3/α4 FMN binding loop 4 contribute to both NADH and metal ion binding, we propose a model in which structural changes around the FMN binding pocket couples to both chromate and NADH binding sites.
Highlights
Chromate [Cr(VI)] is a toxic pollutant that commonly leaches into ground water [1]
NADH and metal ion binding, we propose a model in which structural changes around the FMN binding pocket couples to both chromate and NADH binding sites
One class involves a metal reductase enzyme in the outer membrane that couples through a complex series of specialized electron transfer proteins [4,5,6,7]; the second class involves a cysosolic NAD(P)Hdependent chromate reductase (i.e., ChrR) that functions autonomously [2,8,9]
Summary
Chromate [Cr(VI)] is a toxic pollutant that commonly leaches into ground water [1]. Recently it has been found that some bacteria express enzymes that are able to bind and reduce toxic Cr(VI) to form nontoxic Cr(III) as an intracellular precipitate, offering a cost-effective strategy of bioremediation [2,3]. Following electron transfer and reduction of chromate, NAD(P)+ must dissociate prior to the release of the reduced chromate. Consistent with this latter mechanism, the crystal structure of ChrR (3s2y.pdb) suggests a likely ion binding site for chromate proximal to FMN within the NAD(P)H binding pocket [8]. As chromate reduction by ChrR involves an adventitious enzyme activity [9] with a low catalytic rate and binding affinity (kcat = 0.25 sec−1; Km = 0.24 mM) [8,13], the precise role of the FMN cofactor in chromate reduction remains uncertain. Consistent with the proposal that FMN plays a catalytic role in chromate reduction, we observe substantial decreases in catalytic rates of chromate reduction upon the site-directed mutagenesis of four different FMN ligands (S15A, R17A, E83A, and Y84A) in the FMN binding pocket
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