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

Abstract

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(II) as a Lewis acid under normal growth conditions but which switch to Mn(II) under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe(II) and Mn(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.

Fig. 1

Comparison of the metal binding sites of Fe- and Mn-containing extradiol dioxygenases (A), SODs (B), and the class Ia and Ib RNRs (C), which are the primary subjects of this review. (A) Active sites of the Fe-containing homoprotocatechuate 2,3-dioxygenase (HPCD) of Brevibacterium fuscum (white) (PDB code: 1Q0C) and the Mn-containing HPCD (MndD) of Arthrobacter globiformis (pink, 1F1V), each soaked with homoprotocatechuate (orange). (B) Active sites of the FeSOD (white, 1ISB) and MnSOD (pink, 1D5N) of E. coli. (C) Reduced (top) and oxidized (bottom) forms of the class Ia and Ib RNRs. At the top, the diferrous form of E. coli class Ia RNR (1PIY, white) is overlaid with the dimanganese(^II) form of B. subtilis class Ib RNR (4DR0, chain A, pink). At the bottom, the diferric form of E. coli class Ia RNR (1MXR, white) is overlaid with the dimanganese(III) form of C. ammoniagenes class Ib RNR (3MJO, pink). Mn ions are purple spheres, Fe ions are brown spheres, and solvent molecules are red spheres. Metal–ligand bonds are shown as dotted lines. Figures were generated using PyMOL.

Fig. 2

Reactions catalyzed by the enzymes discussed in this review, where M=Mn or Fe. (A) Ribulose 5-phosphate 3-epimerase (Rpe) catalyzes the reversible interconversion of ribulose 5-phosphate and xylulose 5-phosphate. (B) A representative of the extradiol dioxygenases, homoprotocatechuate 2,3-dioxygenases catalyze the oxidation of homo-protocatechuate to 2-hydroxy-5-carboxymethylmuconate semialdehyde. (C) Superoxide dismutases (SODs) catalyze the disproportionation of O₂^•− to O₂ and H₂O₂. (D) Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside 5′-diphosphates (NDPs) to deoxynucleoside 5′-diphosphates (dNDPs). The class I RNRs use two dimeric subunits, α2 and β2, in a 1 : 1 complex; only one monomer is shown here. The β2 subunit contains the metallocofactor and the α2 subunit contains the site of nucleotide reduction. Upon binding of substrate (NDP, shown) and nucleotide effector (not shown) to α2, Y^• oxidizes a Cys residue in the α2 active site to a thiyl radical (S^•), which initiates nucleotide reduction. Here we show the reaction of a class Ia RNR (M=Fe). The reactions of the class Ib and Ic RNRs are similar, with differences related to their distinct cofactors. In the class Ib RNRs (M = Mn), either O₂^•− or HOO(H) (not yet determined) is the oxidant in cofactor assembly. In the class Ic RNRs, the tyrosine shown for the class Ia and Ib RNRs is replaced by a redox-inert residue (Phe in C. trachomatis RNR) and the active enzyme employs a Mn^IVFe^III cofactor in the β subunit.

Fig. 3

Model for the interrelation of oxidative stress and Fe limitation in expression of the class Ib RNR in E. coli. In this proposal, H₂O₂ can inactivate Lewis acid-requiring enzymes that utilize Fe^II in catalysis by oxidizing Fe^II to Fe^III, which irreversibly dissociates (as a result of ligand modification, for example).^, Fur is also inactivated, presumably by a similar mechanism. Loss of Fe from Fur activates transcription of Fur-repressed genes such as mntH and nrdHIEF, mimicking general Fe limitation.^, H₂O₂ also induces, via OxyR, expression of MntH and the ferritin-like protein Dps. Dps sequesters free iron using H₂O₂ as an oxidant. MntH imports Mn^II. H₂O₂ leads to increased levels of the apo form of IscR, by destroying the [2Fe2S] cluster of IscR (shown here) and/or by interfering with its assembly (as proposed by Imlay); apo-IscR positively regulates nrdHIEF transcription. Together, Mn^II import and Fe^II oxidation and sequestration leads to NrdF being metallated with Mn^II, with the essential oxidant for cluster assembly provided by NrdI’s reaction with O₂.

Fig. 4

Classes of RNRs. RNRs are classified on the basis of the metallocofactor used to reversibly generate the cysteine thiyl radical (red) essential for catalysis. Class Ia RNRs use a diferric-Y^• cofactor, class Ib RNRs use a dimanganese(^III)-Y^• cofactor, class Ic RNRs use a Mn^IVFe^III cofactor, class II RNRs use adenosylcobalamin, and class III RNRs use a glycyl radical generated by an activating enzyme requiring S-adenosylmethionine and a [4Fe4S]⁺ cluster.

Fig. 5

General mechanisms of assembly of the metallocofactors of the class I RNRs. Two proposed mechanisms for Mn^III ₂-Y^• cofactor assembly in class Ib RNRs are shown.^,, The identities of the bridging ligands are only definitively established for the class Ia RNRs.

Fig. 6

The proposed oxidant access route for Mn^III ₂-Y^• cofactor assembly in the E. coli class Ib RNR complex of NrdI (green) and NrdF (gray) (PDB code: 3N3A). Water molecules in the channel connecting the FMN cofactor (yellow) of NrdI and the Mn^II ₂ site (purple spheres) of NrdF are shown in blue spheres and mesh, and the highly conserved hydrophilic residues from NrdI and NrdF lining the channel are indicated.

Fig. 7

The importance of accessory factors in metallation and assembly of the E. coli class Ia and Ib RNR metallocofactors. The structural images were created in PyMOL from the crystal structures of E. coli Fe^III ₂–NrdB (1MXR), C. ammoniagenes Mn^III ₂–NrdF (3MJO), and S. Typhimurium Fe^III ₂–NrdF (2R2F).