Structural Basis for Assembly of the Mn^IV/Fe^III Cofactor in the Class Ic Ribonucleotide Reductase from Chlamydia trachomatis

Abstract

The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) employs a MnIV/FeIII cofactor in each monomer of its β2 subunit to initiate nucleotide reduction. The cofactor forms by reaction of MnII/FeII-β2 with O2. Previously, in vitro cofactor assembly from apo β2 and divalent metal ions produced a mixture of two forms, with Mn in site 1 (MnIV/FeIII) or site 2 (FeIII/MnIV), of which the more active MnIV/FeIII product predominates. Here we have addressed the basis for metal site-selectivity by solving X-ray crystal structures of apo, MnII, and MnII/FeII complexes of Ct β2. A structure obtained anaerobically with equimolar MnII, FeII, and apo protein reveals exclusive incorporation of MnII in site 1 and FeII in site 2, in contrast to the more modest site-selectivity achieved previously. Site-specificity is controlled thermodynamically by the apo protein structure, as only minor adjustments of ligands occur upon metal binding. Additional structures imply that, by itself, MnII binds in either site. Together the structures are consistent with a model for in vitro cofactor assembly in which FeII specificity for site 2 drives assembly of the appropriately configured heterobimetallic center, provided that FeII is substoichiometric. This model suggests that use of an MnIV/FeIII cofactor in vivo could be an adaptation to FeII limitation. A 1.8 Å resolution model of the MnII/FeII-β2 complex reveals additional structural determinants for activation of the cofactor, including a proposed site for side-on (η2) addition of O2 to FeII and a short (3.2 Å) MnII-FeII interionic distance, promoting formation of the MnIV/FeIV activation intermediate.

Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor

“…In class Ia and Ib R2s, helix E adopts a regular ␣-helical structure, and the metal ligand corresponding to Glu-167 shifts to bidentate coordination mode upon reduction (66 -71). As noted above, this shift does not occur in R2c or R2lox (20,22,80) nor in BMMs (72)(73)(74)(75)(76)(77)(78)(79). This difference between class Ia and Ib R2 proteins on the one hand and R2c, R2lox, and BMMs on the other hand correlates with the different nature of their N-terminal metal ligand, an aspartate in the former group and a glutamate in the latter, thus providing the class Ia and Ib R2 proteins with an overall more flexible active site.…”

“…The same single carboxylate shift occurs in the founding member of the other group of Mn/Fe proteins, the Chlamydia trachomatis class Ic R2 protein (R2c) (20,80). The residue corresponding to Glu-167 is a monodentate ligand to the site 2 metal ion in BMMs and Mn/Fe proteins, whereas it shifts from bidentate to monodentate ligation upon oxidation in class Ia and Ib R2 proteins.…”

“…In R2lox, however, it is only slightly larger in the reduced than in the oxidized state (Table 5), as is also the case in BMMs (72-79, 94 -96). Similarly, in R2c the metal-metal distance increases by only 0.1-0.2 Å upon reduction, although the metal ions are much closer to each other in this protein than in R2lox, with a distance of 3.2 Å in the Mn II /Fe II state (20,21,(55)(56)(57)80). It would appear that the active site geometry is more restrained in the Mn/Fe proteins and BMMs than in class Ia and Ib R2 proteins.…”

“…Channels proposed to allow access of the oxidant to the metal center are present in the equivalent position in class Ia and Ib R2 proteins (81-84). In the recently solved structure of R2c in the reduced state, there is no such channel, but the protein surface displays a cavity in the same position as R2lox, with only Ile-231 (corresponding to Ile-206 in R2lox) blocking access to site 2 (80). Thus, the structures of R2lox presented here are in line with proposals made for other di-metal carboxylate proteins that oxygen first binds to site 2 (10, 67, 80 -83, 85).…”

“…9B). Helix E is -helical in both the oxidized and the reduced state in R2lox and R2c (20,22,80), and consequently also in the absence of the ether cross-link, whereas in BMMs, helix E forms a -helix upon binding of the regulatory subunit (78,79). The cross-link in R2lox appears to impose additional strain and probably enforces this rigid geometry during catalysis to prevent unwanted side reactions.…”