An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe-4S] clusters.

The biosynthesis of the organometallic H cluster of [Fe-Fe] hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe-S protein with GTPase activity. The [4Fe-4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX(46-53)HisCysXXCys.

ENDOR spectroscopy shows that guanine N1 binds to [4Fe-4S] cluster II of the S-adenosylmethionine-dependent enzyme MoaA: mechanistic implications.

The S-adenosylmethionine-dependent enzyme MoaA, in concert with MoaC, catalyzes the first step of molybdenum cofactor biosynthesis, the conversion of guanosine 5'-triphosphate (5'-GTP) into precursor Z. A published X-ray crystal structure of MoaA with the substrate 5'-GTP revealed that the substrate might be bound to the unique iron of one of two 4Fe-4S clusters through either or both the amino and N1 nitrogen nuclei. Use of 35 GHz continuous-wave ENDOR spectroscopy of MoaA with unlabeled and (15)N-labeled substrate and a reduced [4Fe-4S](+) cluster now demonstrates that only one nitrogen nucleus is bound to the cluster.

MiaB, a bifunctional radical-S-adenosylmethionine enzyme involved in the thiolation and methylation of tRNA, contains two essential [4Fe-4S] clusters.

The radical-S-adenosylmethionine (radical-AdoMet) enzyme MiaB catalyzes the posttranscriptional methylthiolation of N-6-isopentenyladenosine in tRNAs. Spectroscopic and analytical studies of the reconstituted wild-type and C150/154/157A triple variant forms of Thermotoga maritima MiaB have revealed the presence of two distinct [4Fe-4S](2+,1+) clusters in the protein. One is coordinated by the three conserved cysteines in the radical-AdoMet motif (Cys150, Cys154, and Cys157) as previously reported, and the other, here observed for the first time, is proposed to be coordinated by the three N-terminal conserved cysteines (Cys10, Cys46, and Cys79).

Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis.

The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus.

Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase.

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe.

Characterization of the cofactor composition of Escherichia coli biotin synthase.

The cofactor content of in vivo, as-isolated, and reconstituted forms of recombinant Escherichia coli biotin synthase (BioB) has been investigated using the combination of UV-visible absorption, resonance Raman, and Mössbauer spectroscopies along with parallel analytical and activity assays. In contrast to the recent report that E. coli BioB is a pyridoxal phosphate (PLP)-dependent enzyme with intrinsic cysteine desulfurase activity (Ollagnier-deChoudens, S.

MiaB protein from Thermotoga maritima. Characterization of an extremely thermophilic tRNA-methylthiotransferase.

In Escherichia coli, the MiaB protein catalyzes the methylthiolation of N-6-isopentenyl adenosine in tRNAs, the last reaction step during biosynthesis of 2-methylthio-N-6-isopentenyl adenosine (ms2i6A-37). For the first time the thermophilic bacterium Thermotoga maritima is shown here to contain such a MiaB tRNA-modifying enzyme, named MiaBTm, and to synthesize ms2i6A-37 as demonstrated by an analysis of modified nucleosides from tRNA hydrolysates. The corresponding gene (TM0653) was identified by sequence similarity to the miaB gene cloned and expressed in E.