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 one-electron 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) (kcat/Km = approximately 2 x 10(4) M(-1) x 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.

FIG. 1.

Clustal W alignment of YieF and ChrR. The asterisks indicate identical residues, the colons indicate residues with a high level of similarity, and the periods indicate residues with a lower level of similarity. The characteristic signature of the NADH_dh2 family of proteins is boxed, and the 18 residues that are particularly highly conserved within ChrR homologs are shaded.

FIG. 2.

Rapid-mixing studies with ChrR and YieF. The left column presents data for the ChrR protein; the right column presents data for the YieF protein. (A) Spectra extracted from the data sets collected when ChrR or YieF (3 μM) was rapidly mixed with limiting NADH (10 μM) and an excess of chromate (40 μM). The spectra were obtained from a global fit of all the absorbance-versus-wavelength-versus-time data to a biphasic kinetic mechanism. Spectrum a, reduced enzyme; spectrum b, semiquinone form of FMN; spectrum c, oxidized enzyme. (B to D) Time courses of absorbance changes at A₅₀₁ (B, showing the disappearance of the reduced enzyme), A₄₀₃ (C, showing the appearance of the oxidized enzyme), and A₅₈₀ (D, showing flavin semiquinone formation) when oxidized ChrR or YieF was mixed with NADH and different molar excesses of chromate. The final concentrations after mixing were as follows: ChrR, 3.0 μM; NADH, 10 μM; chromate, 20 (trace a) or 40 (trace b) μM.

FIG. 3.

(A) Growth of wild-type P. putida KT2440 in LB medium with (▪) or without (•) 400 μM Cr(VI). (B) Growth of wild-type P. putida KT2440 (▪) and the CRK4 mutant (▴) in LB medium with 400 μM Cr(VI). The data are the mean points of three independent measurements; the standard error of the mean was <7%.

FIG. 4.

Initial rates of chromate removal from LB medium by wild-type (KT2440; •) and mutant (CRK4; ▪) cell suspensions. The results are averages of five independent measurements; the standard error of the mean was <5%.

FIG. 5.

Relative band intensities of Western immunoblot detection of ChrR in P. putida KT2440 grown in LB medium with (▪) or without (•) 400 μM Cr(VI). The 8-h (darkest) band intensity, seen in cultures containing chromate, was taken as 100%. The inset shows Western bands for the challenged and unchallenged samples. The numbers indicate the collection time points in hours; 26 μg of total protein was loaded in each lane. Qualitatively similar results were seen in two independent measurements.

FIG. 6.

β-Galactosidase activity determined during growth of AMS-6λY in LB medium without (•) or with (▪) 200 μM chromate. The open symbols show culture growth monitored at A₆₆₀ in the absence (○) or presence (□) of chromate. The results represent averages of three experiments; the standard error of the mean was <5%.