Abstract
The FS0 [4Fe-4S] cluster of the catalytic subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase (DmsABC) plays a key role in the electron transfer relay. We have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. EPR spectroscopy indicates that FS0 has a high spin ground state (S = 3/2) in its reduced form, resulting in an EPR spectrum with a peak at g ∼ 5.0. The cluster is predicted to be in close proximity to the molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor, which provides the site of dimethyl sulfoxide reduction. Comparison with nitrate reductase A (NarGHI) indicates that a sequence of residues ((18)CTVNC(22)) plays a role in both FS0 and Mo-bisPGD coordination. A DmsA(ΔN21) mutant prevented Mo-bisPGD binding and resulted in a degenerate [3Fe-4S] cluster form of FS0 being assembled. DmsA belongs to the Type II subclass of Mo-bisPGD-containing catalytic subunits that is distinguished from the Type I subclass by having three rather than two residues between the first two Cys residues coordinating FS0 and a conserved Arg residue rather than a Lys residue following the fourth cluster coordinating Cys. We introduced a Type I Cys group into DmsA in two stages. We changed its sequence from (18)C(A)TVNC(B)GSRC(C)P(27) to (18)C(A)TYC(B)GVGC(C)G(26) (similar to that of formate dehydrogenase (FdnG)) and demonstrated that this eliminated both Mo-bisPGD binding and EPR-detectable FS0. We then combined this change with a DmsA(R61K) mutation and demonstrated that this additional change partially rescued Mo-bisPGD insertion.
Highlights
Grant MOP15292. 1 Supported by a Natural Sciences and Engineering Research Council of Canada graduate studentship and a Queen Elizabeth II graduate scholarship. 2 To whom correspondence should be addressed: Dept. of Biochemistry, 474 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G2H7,Canada.Tel.:780-492-2761;Fax:780-492-0886;E-mail:jweiner@ ualberta.ca
Each structurally characterized enzyme of this family consists of a catalytic subunit with a molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD)3 cofactor and an FS0 [4Fe-4S] cluster, an electron transfer subunit containing four [Fe-S] clusters, and a membrane anchor subunit containing a quinol/quinone-binding site (Q-site)
The third residue after CA in DmsA is an Asn, which is highly conserved in DmsABC-type dimethyl sulfoxide reductases and NarGHI-type nitrate reductases across bacterial species [2]
Summary
Grant MOP15292. 1 Supported by a Natural Sciences and Engineering Research Council of Canada graduate studentship and a Queen Elizabeth II graduate scholarship. 2 To whom correspondence should be addressed: Dept. of Biochemistry, 474 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G2H7,Canada.Tel.:780-492-2761;Fax:780-492-0886;E-mail:jweiner@ ualberta.ca. In E. coli DmsABC, it is the DmsA catalytic subunit that contains the Mo-bisPGD cofactor and an FS0 [4Fe-4S] cluster. An important distinction between the Type II Cys group sequence of DmsA and that of NarG is that there is a His in the CA position of the latter and a Cys at this position in the former It was not known whether the observation of a high spin FS0 EPR spectrum is related to the presence of His at the CA position.
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