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[superscript III][subscript 2]-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe[superscript II][subscript 2]-NrdB, O[subscript 2], and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn[superscript III][subscript 2]-Y• cofactor in their NrdF subunit. Mn[superscript II][subscript 2]-NrdF does not react with O[subscript 2], but it binds the reduced form of a conserved flavodoxin-like protein, NrdI[subscript hq], which, in the presence of O[subscript 2], reacts to form the Mn[superscript III][subscript 2]-Y• cofactor. Here we investigate the mechanism of assembly of the Mn[superscript III][subscript 2]-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn[superscript II][subscript 2]-NrdF, NrdI[subscript hq], and O[subscript 2] has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI[subscript hq] reduces O[subscript 2] to O[subscript 2]•– (40–48 s[superscript –1], 0.6 mM O[subscript 2]), the O[subscript 2]•– channels to and reacts with Mn[superscript II][subscript 2]-NrdF to form a Mn[superscript III]Mn[superscript IV] intermediate (2.2 ± 0.4 s[superscript –1]), and the Mn[superscript III]Mn[superscript IV] species oxidizes tyrosine to Y• (0.08–0.15 s[superscript –1]). Controlled production of O[subscript 2]•– by NrdI[subscript hq] during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn[superscript II][subscript 2] cluster with O[subscript 2] and satisfies the requirement for an “extra” reducing equivalent in Y• generation.