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

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.National Institutes of Health (U.S.) (Grant GM81393)United States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships

O₂-Evolving Chlorite Dismutase as a Tool for Studying O₂-Utilizing Enzymes

The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas’s modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, <i>in situ</i> generation of O₂ at concentrations far exceeding 2 mM. This method was used to define the O₂ concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from <i>Chlamydia trachomatis</i>, in which the enzyme’s Mn^IV/Fe^III cofactor forms from a Mn^II/Fe^II complex and O₂ via a Mn^IV/Fe^IV intermediate, at effective O₂ concentrations as high as ∼10 mM. With a more soluble receptor, myoglobin, an O₂ adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C–H-bond-cleaving Fe^IV–oxo complex, <b>J</b>, in taurine:α-ketoglutarate dioxygenase and superoxo–Fe₂^III/III complex, <b>G</b>, in <i>myo</i>-inositol oxygenase, and the tyrosyl-radical-generating Fe₂^III/IV intermediate, <b>X</b>, in <i>Escherichia coli</i> RNR, were all accumulated to yields more than twice those previously attained. This means of <i>in situ</i> O₂ evolution permits a >5 mM “pulse” of O₂ to be generated in <1 ms at the easily accessible Cld concentration of 50 μM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O₂ addition steps