A Manganese(IV)/Iron(III) Cofactor in
            <i>Chlamydia trachomatis</i>
            Ribonucleotide Reductase

Jiang, Wei; Yun, Danny; Saleh, Lana; Barr, Eric W.; Xing, Gang; Hoffart, Lee M.; Maslak, Monique Anne; Krebs, Carsten; Bollinger, J. Martin

doi:10.1126/science.1141179

Cited by 187 publications

(406 citation statements)

References 24 publications

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“…The major doublet (75% intensity) arises from the functional Mn IV /Fe III cofactor. 11 For in-depth characterization of this species, we collected Mössbauer spectra at 4.2 K in variable applied magnetic fields. To analyze these spectra, it was necessary first to remove the 25% contribution attributed to the Fe 2 III/III complex ( Figure S1).…”

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confidence: 99%

“…The metal cluster undergoes reduction from Mn IV /Fe III to Mn III /Fe III , which is, by contrast to the reduced cofactor of a conventional class I RNR, EPR active. 11 Moreover, although the reduced form might, as in E. coli RNR, not accumulate during turnover, it can be prepared in stable form and its structure is affected by binding of R1 and nucleotides, as demonstrated by marked changes to its EPR spectrum. 11 The structural changes caused by formation of the complex are almost certainly elements of the gating mechanism.…”

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confidence: 99%

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The Active Form of Chlamydia trachomatis Ribonucleotide Reductase R2 Protein Contains a Heterodinuclear Mn(IV)/Fe(III) Cluster with S = 1 Ground State

2007

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The class I ribonucleotide reductase from Chlamydia trachomatis uses a stable Mn IV /Fe III cofactor to initiate nucleotide reduction by a free-radical mechanism. The enzyme provides the first example both of a Mn-dependent ribonucleotide reductase and of a Mn/Fe redox cofactor. In this work, we have used variable-field Mössbauer spectroscopy to demonstrate that the active cofactor has an S = 1 ground state due to antiferromagnetic coupling between the Mn IV (S Mn = 3/2) and high-spin Fe III (S Fe = 5/2) sites.The reduction of nucleotides to 2′-deoxynucleotides by ribonucleotide reductases (RNRs) 1 begins with the abstraction of the 3′ hydrogen atom of the substrate by a transient cysteine thiyl radical (C•). [2][3][4] Class I, II, and III RNRs differ in subunit composition and the nature of the cofactor used to generate the 3′-H-abstracting C•. 2 A conventional class I RNR uses a stable tyrosyl radical (Y•), which is introduced into the enzyme's homodimeric R2 subunit (also denoted β 2 ) by reaction of O 2 with an adjacent carboxylate-bridged non-heme diiron cluster, 5 to generate the C• in its R1 subunit (also denoted α 2 ) via long-range (~ 35 Å) proton-coupled electron transfer (PCET). [6][7][8] The identification in Chlamydia trachomatis (Ct) and other species of pathogenic bacteria of RNRs having the class I subunit architecture but the Y•-harboring tyrosine replaced by phenylalanine raised the question of how such an RNR might function without the initiating Y•. 9,10 We recently showed that Ct RNR uses a stable Mn IV /Fe III cofactor, generated by reaction of the Mn II /Fe II -R2 complex with O 2 , for radical initiation. 11 Although heterobinuclear Mn/Fe complexes of various oxidation states, 12-14 including one example of an inorganic Mn IV /Fe III complex, 15 have been reported, Ct RNR is, to our knowledge, the first case in which an enzyme has been shown to use a Mn/Fe cluster as a redox cofactor. Here we characterize this novel cofactor by Mössbauer spectroscopy, showing that it has a triplet (S Total = 1) ground state resulting from antiferromagnetic coupling of its Mn IV (S Mn = 3/2) and high-spin Fe III (S Fe = 5/2) constituents and providing parameters to calibrate calculation of its geometric and electronic structure. Figure 2, analysis of these spectra for the hypothetical case of an S Total = 4 ground state, selected plots of the spin expectation values, and the spin Hamiltonian used for analysis of the Mössbauer spectra. This material is available free of charge from the journal website: http://pubs.acs.org. Figure S1). The derived spectra of the Mn IV / Fe III complex are shown in Figure 2. NIH Public AccessThe zero-field spectrum is a sharp quadrupole doublet with parameters (δ = 0.52 mm/s and ΔE Q = 1.32 mm/s) typical of high-spin Fe III . Spectra recorded in applied fields were analyzed with the assumption of slow relaxation and according to the spin Hamiltonian with respect to the total-spin ground state, S Total = 1, given in the Supporting Information. For this analysis, the ...

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The Active Form of Chlamydia trachomatis Ribonucleotide Reductase R2 Protein Contains a Heterodinuclear Mn(IV)/Fe(III) Cluster with S = 1 Ground State

2007

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“…However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7)(8)(9)(10)(11)(12)(13)(14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn IV /Fe III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9,10). After a long controversy, class Ib R2 proteins were shown to use a dimanganese cofactor in the same scaffold (7,8).…”

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confidence: 99%

“…For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7)(8)(9)(10)(11)(12)(13)(14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn IV /Fe III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10).…”

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Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Griese¹,

Roos

Cox

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

283

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Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn II < Fe II < Ni II < Co II < Cu II > Zn II ). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn II and Fe II in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe II over Mn II as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a twostep process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.H alf of all enzymes are estimated to contain metallocofactors (1). An important subset uses transition metal ions to perform key redox reactions such as oxygen activation. The diiron cofactor of the ferritin-like superfamily of proteins is particularly versatile (2). While ferritin itself simply oxidizes and sequesters iron (3), in other family members the diiron center acts as a transient one-or two-electron oxidant. In the R2 subunits of class I ribonucleotide reductases (RNRs) it generates a redoxactive tyrosyl radical (4, 5), whereas in the bacterial multicomponent monooxygenases (BMMs) it catalyzes the hydroxylation of a variety of hydrocarbons (6). For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7)(8)(9)(10)(11)(12)(13)(14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn IV /Fe III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10). After a long controversy, class Ib R2 proteins were shown to use a dimanganese cofactor in the same scaffold (7,8). These recent developments highlight the complexity of correctly identifying the metals that make up native metallocofactors. While the metal preferences of some primary coordination motifs are well known and distinct, others ar...

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“…The involvement of a metal (M) other than Fe in vivo has not been ruled out in the case of NrdF. M may be Fe (13), Mn (39), or Mn/Fe (40). O 2 and an extra electron have been shown to be required for cluster assembly of NrdB.…”

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confidence: 99%

NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase

Cotruvo¹,

Stubbe

2008

Proc. Natl. Acad. Sci. U.S.A.

125

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(Fig. 1). Our studies have demonstrated the importance in E. coli of a [2Fe2S]-ferredoxin (Fd), YfaE, in the class Ia maintenance and possibly biosynthetic pathways (3, 4). We now report that NrdI is a flavodoxin (Fld), functioning as a two-electron reductant of NrdF (␤2), and can play a role in the maintenance pathway of the E. coli class Ib RNR analogous to that of YfaE in the E. coli class Ia system. E. coli contains genes for three RNRs that have been classified based on their metallocofactors: two class I RNRs (Ia and Ib) and a class III RNR. The class Ia RNR is the workhorse enzyme and is expressed under normal aerobic, vegetative growth conditions, whereas the class Ib enzyme is expressed under oxidative stress and iron-limited growth conditions (5-8). The class III RNR is expressed only under anaerobic conditions and will not be discussed further. The E. coli class Ia RNR is composed of two gene products, NrdA (␣) and NrdB (␤), and the genes are found in an operon with a [2Fe2S]-Fd, YfaE. The E. coli class Ib RNR is composed of NrdE (␣) and NrdF (␤) and the genes are found in an operon (nrdHIEF) with two additional genes. NrdH is a thioredoxin-like protein that functions as the specific disulfide reductase for NrdE (9). NrdI has yet to be functionally characterized. However, NrdI is annotated in the genomic databases as a Fld and a recent structure of the Bacillus subtilis NrdI . O 2 and an extra electron have been shown to be required for cluster assembly of NrdB. The substoichiometric assembly of cluster in NrdF suggests further investigation of cluster assembly is required. Fre may play an important role in the maintenance pathway and may be a reductase for YfaE (4, 41).

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A Manganese(IV)/Iron(III) Cofactor in Chlamydia trachomatis Ribonucleotide Reductase

Cited by 187 publications

References 24 publications

The Active Form of Chlamydia trachomatis Ribonucleotide Reductase R2 Protein Contains a Heterodinuclear Mn(IV)/Fe(III) Cluster with S = 1 Ground State

The Active Form of Chlamydia trachomatis Ribonucleotide Reductase R2 Protein Contains a Heterodinuclear Mn(IV)/Fe(III) Cluster with S = 1 Ground State

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

NrdI, a flavodoxin involved in maintenance of the diferric-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase

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