title: Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase: enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate.
abstract: Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y(•)) generated by oxidation of a reduced dinuclear metal cluster. The Fe(III)2-Y(•) cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe(II)2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn(III)2-Y(•) cofactor in their NrdF subunit. Mn(II)2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the Mn(III)2-Y(•) cofactor. Here we investigate the mechanism of assembly of the Mn(III)2-Y(•) cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn(II)2-NrdF, NrdI(hq), and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI(hq) reduces O2 to O2(•-) (40-48 s(-1), 0.6 mM O2), the O2(•-) channels to and reacts with Mn(II)2-NrdF to form a Mn(III)Mn(IV) intermediate (2.2 ± 0.4 s(-1)), and the Mn(III)Mn(IV) species oxidizes tyrosine to Y(•) (0.08-0.15 s(-1)). Controlled production of O2(•-) by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn(II)2 cluster with O2 and satisfies the requirement for an "extra" reducing equivalent in Y(•) generation.
authors: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors: Joseph A
authors: JA
authors: Cotruvo
authors: Troy A
authors: TA
authors: Stich
authors: R David
authors: RD
authors: Britt
authors: JoAnne
authors: J
authors: Stubbe
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000378:metabolism*; D013481:Superoxides / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D013481:Superoxides; D014443:Tyrosine; D008345:Manganese; D012264:Ribonucleotide Reductases
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