Dataset Information

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.

ABSTRACT: Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.

SUBMITTER: Hamann N

PROVIDER: S-EPMC3543786 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.

Hamann Nils N Mander Gerd J GJ Shokes Jacob E JE Scott Robert A RA Bennati Marina M Hedderich Reiner R

Biochemistry 20071012 44

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cy ...[more]

PMID: 17929940

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Project description:Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5'-deoxyadenosyl 5'-radical that is generated by a reductive cleavage of S-adenosyl- l-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in iron-sulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 +/- 0.4 irons and 12.2 +/- 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S] (2+) configuration, as determined by Mossbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 +/- 0.2 irons and 9.9 +/- 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mossbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys --> Ala substitutions at each of the cysteines in its CX 3CX 2C radical SAM motif contained 7.3 +/- 0.1 irons and 7.2 +/- 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 +/- 0.1 irons and 8.4 +/- 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV-visible and Mossbauer spectra of both samples indicated the presence of only [4Fe-4S] clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a V max/[E T] of approximately 0.36 min (-1) for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of approximately 0.039 min (-1) for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5'-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is changed to cysteine also gives rise to turnover, supporting approximately 4-fold the activity of that observed with the natural substrate.

Dataset Information

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.

Publications

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.

Similar Datasets

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