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

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use Fe[superscript II] as a Lewis acid under normal growth conditions but which switch to Mn[superscript II] under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe[superscript II] and Mn[superscript II], the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.National Institutes of Health (U.S.) (Grant GM81393

Multiply Resistant (MR) Salmonella enterica Serotype Typhimurium DT 12 and DT 120: A Case of MR DT 104 in Disguise?

Multiresistant Salmonella enterica serotype Typhimurium definitive phage type (DT) 12 and DT 120 are more closely related to DT 104 than to non-multiresistant strains of their respective phage types. Multiresistant DT 12 and DT 120 appear to have arisen due to changes in phage susceptibility of DT 104 rather than horizontal transfer of resistance genes

Direct Measurement of the Radical Translocation Distance in the Class I Ribonucleotide Reductase from <i>Chlamydia trachomatis</i>

Ribonucleotide reductases (RNRs) catalyze conversion of ribonucleotides to deoxyribonucleotides in all organisms via a free-radical mechanism that is essentially conserved. In class I RNRs, the reaction is initiated and terminated by radical translocation (RT) between the α and β subunits. In the class Ic RNR from <i>Chlamydia trachomatis</i> (<i>Ct</i> RNR), the initiating event converts the active <i>S</i> = 1 Mn­(IV)/Fe­(III) cofactor to the <i>S</i> = 1/2 Mn(III)/Fe(III) “RT-product” form in the β subunit and generates a cysteinyl radical in the α active site. The radical can be trapped via the well-described decomposition reaction of the mechanism-based inactivator, 2′-azido-2′-deoxyuridine-5′-diphosphate, resulting in the generation of a long-lived, nitrogen-centered radical (N^•) in α. In this work, we have determined the distance between the Mn­(III)/Fe­(III) cofactor in β and N^• in α to be 43 ± 1 Å by using double electron–electron resonance experiments. This study provides the first structural data on the <i>Ct</i> RNR holoenzyme complex and the first direct experimental measurement of the inter-subunit RT distance in any class I RNR

Evidence That the β Subunit of <i>Chlamydia trachomatis</i> Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the β subunit oxidizes a cysteine residue ∼35 Å away in the α subunit, generating a thiyl radical. In the class Ic enzyme from <i>Chlamydia trachomatis</i> (<i>Ct</i>), the cysteine oxidant is the Mn^IV ion of a Mn^IV/Fe^III cluster, which assembles in a reaction between O₂ and the Mn^II/Fe^II complex of β. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn^IV ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn^IV ion most likely resides in this site (i.e., ¹Mn^IV/²Fe^III), but a subsequent computational study favored its occupation of site 2 (¹Fe^III/²Mn^IV). In this work, we have sought to resolve the location of the Mn^IV ion in <i>Ct</i> RNR-β by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted <i>in vitro</i> by two different procedures. In samples containing primarily Mn^IV/Fe^III clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both ¹Mn^II/²Fe^II and ¹Fe^II/²Mn^II complexes are competent to react with O₂ to produce the corresponding oxidized states. However, with diminished Mn^II loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these “low-Mn” samples on a per-Mn basis implies that the ¹Mn^IV/²Fe^III-β is at least the more active of the two oxidized forms and may be the only active form