Chromate-Reducing Properties of Soluble Flavoproteins from Pseudomonas putida and Escherichia coli

Abstract

Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze oneelectron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins-ChrR (from Pseudomonas putida) and YieF (from Escherichia coli)-were examined. Both are dimers and reduce chromate efficiently to Cr(III) (k cat /K m ‫؍‬ ϳ2 ؋ 10 4 M ؊1 ⅐ s ؊1 ). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

“…ecAzoR, rsAzoR, stAzoR and efAzoR are azoreductases from E. coli (Nakanishi et al, 2001), R. sphaeroides (Bin et al, 2004), S. typhimurium (Zhang et al, 2004) and E. faecalis (Chen et al, 2004). paWrbA is the sequence for WrbA from P. aeruginosa (Gorman and Shapiro, 2005), KefF is an enzyme from E. coli (Roosild et al, 2009), ecMdaB and paMdaB are the mediator of drug activity B from E. coli (Adams and Jia, 2006) and P. aeruginosa respectively, YhdA is an enzyme from B. subtilis (Binter et al, 2009), EmoB is an enzyme fromMesorhizobiums p. BNC1 (Nissen et al, 2008), ArsH is an enzyme from S. meliloti (Ye et al, 2007), Lot6p is an enzyme from S. cerevisiae (Liger et al, 2004), ppChrR is the chromate reductase from P. putida (Ackerley et al, 2004). 1X77 is an NADPH dependent oxidoreductase from P. aeruginosa (Agarwal et al, 2006), hNQO1 and hNQO2 are human NQOs (Foster et al, 1999;Li et al, 1995), A. tha is the sequence for an NAD(P)H quinone reductase from A. thaliana (Sparla et al, 1999).…”

In Situ Long-Term Reductive Bioimmobilization of Cr(VI) in Groundwater Using Hydrogen Release Compound

“…As an example, Figure S3 shows the dynamics of Desulfovibrio vulgaris, Geobacter metallireducens, and methanogenic archaea in water samples. The occurrence of these bacteria and archaea suggest a possibility of both direct enzymatically mediated microbial Cr(VI) reduction and an indirect Cr(VI) reduction through byproducts of microbial metabolism such as Fe 2+ and reactive sulfides (21,22,38,39).…”

Extra-cellular chromate-reducing activity of the yeast cultures

“…Anaerobic bacteria can reduce chromate using hydrogenase [6], cytochrome c-dependent electron transfer chains [7,8]. Little is known about mechanisms of chromate reduction for aerobic bacteria, except some enzymes which are shown to catalyze this reaction in vitro, for example, nitrate reductase [9], flavin reductase [10], ferrireductase [11], and some flavoproteins [12,13]. Among bacterial chromate-reducing enzymes are NADH and NAD(P)H-dependent reductases [14].…”