The wild type ChrR crystallized as a tetramer; size exclusion chromatography under conditions used to measure chromate reductase activity revealed this oligomeric form for the active enzyme as well as the mutants listed in ; and manipulation of the amino acid constituents at the dimer-dimer interface markedly improved the ChrR chromate reductase activity. Taken collectively, these findings point to a central role for the tetrameric configuration in chromate reductase activity of this enzyme. Tetrameric association may be essential for the activity of small flavoproteins like ChrR to accommodate their substrates.
Any definitive conclusions as to why the mutants shown in were improved in chromate reductase activity must await co-crystallization of the wild type and the mutant enzymes with chromate and NADH. It is, however, useful for future studies to consider the potential mechanisms that may contribute to this improvement. The substitution of Tyr128 by Asn (ChrR21) is likely to have the following two effects. First, whereas in the wild type enzyme, three of the four hydrogen bonds between the dimers converge on a single oxygen atom of Tyr128 (), Asn substitution would result in replacing them by individual bonds between separate appropriate donor and acceptor atoms [NE(R125)-H…ND2(Asn128), NH1(Arg125)-H…OD1(Asn128) and NH1(R125)-H…OE2(Glu146)] (). Coupled with the fact that Asn is less bulky, the result is likely to be greater stability of the tetramer. A second potential effect concerns the cavity surrounding FMN. While in the native enzyme the Arg125 side chain reaches the solvent and restricts this cavity (), the smaller size of Asn compared to Tyr would make the Arg side chain to turn inward to reach it, resulting in enlarged cavity surrounding FMN and enhancing FMN accessibility (). Similar effects may contribute to the improved activity of the other mutant enzymes, in which also an original bulkier amino acid was replaced by a smaller one. Thus, in ChrR41, replacement of Glu146 by Thr would confer freedom of movement on the side chains of Tyr85 and Ty128, causing the side chain of Arg125 to move towards the other dimer, which would likely make the tetramer more stable and increase the accessible area of FMN. And in ChrR43, the smaller side chain of Met compared to Arg would enlarge the FMN cavity as in ChrR21 ().
Diagramatic representation of the possible ways by which Tyr128Asn substitution in ChrR may increase chromate reductase activity.
Additional factors are also likely to be involved. Tyr128 in the native enzyme is in indirect hydrogen bonding interaction with the FMN isoalloxazine N3H group, through Glu82 and Arg125 side chains in the wild type enzyme (). Its substitution by the shorter chain Asn may alter the hydrogen bonding interactions involving the side chain of Glu82, affecting in turn its interaction with ring III N3H group of FMN. The resulting change in the properties of FMN could therefore also have a role in modifying chromate reductase activity. Similarly, substitution of Arg125 by Met in ChrR43 can be expected to perturb Glu82 properties and hence properties of the FMN ring III.
Regardless, the results presented here indicate a critical role for the hydrogen bond network involving the four amino acids at the tetramer interface (Tyr128, Glu46, Arg125, Tyr85) in chromate reductase activity of ChrR. We have shown that ChrR21 retains its property for chromate reduction without Cr(V) formation 
; it is likely that the other mutants of also retain this character.
Structurally, ChrR belongs to the FMN reductase family of flavodoxin superfamily. Given its physiological role, it is part of the quinone reductase enzymes sharing the flavodoxin-like fold 
. This role is to catalyze quinone reduction by simultaneous two-electron transfer (avoiding formation of the reactive semiquinone intermediate), preempt reduction of diverse electrophiles (e.g., quinones and chromate) by the cellular one-electron reducers and generation of quinols that promote tolerance to ROS 
. It thus constitutes, like the E. coli
WrbA protein, a bridge between bacterial and eukaryotic NAD(P)H:quinone oxidoreductases 
A search for protein data bases showed that ChrR bears the closest structural similarity to the putative FMN-reductase of P. aeruginosa
PA01 T1501 (PDB ID: 1RTT) 
(root mean square deviation, 1.1 Å for 174 atoms; 46% amino acid identity) – the protein used to solve ChrR structure, as noted. In the tetrameric form, T1501 also resembles ChrR in hydrogen bonding interactions at the dimer-dimer interface. Other tetrameric flavoproteins, such as ArsH or the quinone reductase YhdA, while bearing less than 25% sequence similarity with ChrR, also structurally resemble it (Z scores, 22.2 and 21.9, respectively). However, their intermolecular interactions are different. In ChrR, as stated, the dimer-dimer interaction involves a network of hydrogen bonds involving FMN, Glu82, Tyr85 and Arg125 of one dimer, and Tyr128 and Glu146 of the second dimer (). In contrast, the dimer-dimer interaction in YhdA and ArsH involves salt bridges between Lys109 and Asp137 
, and extra loops from N- and C- terminal extensions, respectively 
. Whether these differences affect the substrate range of these enzymes remains to be determined.
One of our applied goals in the work on ChrR has been to generate engineered bacteria for superior bioremediation including their use at contaminated sites. Currently, there is reluctance to introducing engineered bacteria into the environment. As this may change, further improvement in bacterial capacity for in situ
bioremediation by molecular engineering remains a worthwhile approach. In the meantime, however, the results reported here could already lead to improved in situ
bioremediation for the following reason. We have seen that changes in four different amino acids individually caused a significant increase in chromate reductase activity and have shown that at least one of them (ChrR21) 
retains the capacity for ‘safe’ chromate reduction, i.e., without Cr(V) generation. This suggests a strong possibility that bacteria containing such mutant enzymes may have naturally evolved in chromate contaminated sites to escape its toxicity. Isolation of such bacteria from the polluted sites and their use in bioaugmentation of these sites can thus be a promising approach to improve chromate bioremediation 
The atomic coordinates and structure factors (code 3SVL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers.