Class Id ribonucleotide reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes on metal loading

Abstract

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O2·−). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.Electronic supplementary materialThe online version of this article (10.1007/s00775-019-01697-8) contains supplementary material, which is available to authorized users.

The periodic table of ribonucleotide reductases

“…The helical distortion occurs immediately after Mn1 ligand Glu67 ( F. johnsoniae numbering), exposing a ∼300 Å 3 cavity and the metal cluster to solvent ( 14 ). This distortion and cavity have also been observed in metal ion-bound structures of L. blandensis and F. ignava β ( 76 , 77 ). In the former system, the structural deviation seems to be the result of metal ion loading because the corresponding helix is uninterrupted in apo structures of L. blandensis β ( 77 ).…”

“…The oxidized Mn 2 III/IV cluster serves as the radical initiator in this system, making Id enzymes the second example of a metal ion-based Cys oxidant after the heterodimeric Mn IV Fe III cofactor of Ic RNR ( 14 ). Two other class Id RNR β subunits from Facklamia ignava and Leeuwenhoekiella blandensis have been characterized in their activated forms by EPR, yielding similar spectral features to those of Mn 2 III/IV F. johnsoniae β ( 76 , 77 )…”

Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy

“…The L. blandensis NrdB (LbNrdB) was also found to belong to a family of NrdBs with a novel dimanganese metal center that does not use a tyrosyl radical. A high-valent Mn 2 (III,IV) radical is instead generated directly at the metal center, as shown both for LbNrdB the closely related Facklamia ignava NrdB (FiNrdB) (Rozman Grinberg et al, 2018a;Rozman Grinberg et al, 2019).…”

The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron

“…In R2c and R2d subclasses, on the other hand, the radical equivalent is housed on the metal cofactor itself. R2c proteins generate a Mn IV /Fe III cofactor [17][18][19] and the recently discovered class R2d-a Mn IV /Mn III cofactor [10,20,21]. These dimetal centers reside in a ferritin-like scaffold, and are coordinated by two histidine residues and four carboxylate ligands.…”

“…In contrast to the di-Fe II cofactor, di-Mn II is unreactive towards O 2 directly and requires a superoxide species, provided by an FMN coenzyme-containing activase, NrdI [16]. R2d proteins, which also utilize Mn II , scavenge the superoxide from solution and do not require NrdI [20,21]. Heterodinuclear R2c and R2lox proteins, in turn, are capable of reducing O 2 directly via a proposed Mn IV /Fe IV intermediate without any auxiliary factors [4,[24][25][26][27][28].…”