Branched Activation- and Catalysis-Specific Pathways for Electron Relay to the Manganese/Iron Cofactor in Ribonucleotide Reductase from <i>Chlamydia trachomatis</i>

Jiang, Wei; Saleh, Lana; Barr, Eric W.; Xie, Jiajia; Gardner, Monique Maslak; Krebs, Carsten; Bollinger, J. Martin

doi:10.1021/bi800881m

Cited by 46 publications

(85 citation statements)

References 57 publications

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“…Computational studies also suggest that it is capable of oxidizing larger exergonically bound substrates, and indicate that the Mn IV /Fe IV state is more stable than the Fe IV /Fe IV state (34). In line with these calculations, in the Chlamydia trachomatis class Ic R2 protein the Mn IV /Fe IV state has been experimentally observed, whereas it has not been possible to trap the Fe IV /Fe IV state (28,(35)(36)(37)(38)(39). The rationale for using the more complex heterodinuclear cofactor to perform functions that a diiron cofactor is also capable of might therefore be its greater stability.…”

Section: Discussionsupporting

confidence: 52%

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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Section: Discussionsupporting

confidence: 52%

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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“…8, we tentatively summarize the information from our XAS and EPR data, also taking into account previous crystallographic (26,31) and spectroscopic results (27,29,38,48,72). A potential role of the Mn(III)Fe(III) state in oxidation as well as reduction of the metal cluster is suggested.…”

Section: Mn-fe Electronicmentioning

confidence: 65%

“…One additional protonated bridge (OH) in Mn(IV)Fe(III) can explain the elongation of the Mn-Fe distance from ϳ2.75 Å (this study) to ϳ2.9 Å (48). The formation of several Mn(IV)Fe(III) intermediates also has been suggested in a recent Mössbauer investigation on Ct R2 (29) and related to redox transitions of amino acids (Trp-51 andTyr-222) and protolytic reactions. These results point to the presence of several configurations of the active Mn(IV)Fe(III) state, which all could be of functional relevance.…”

Section: Mn-fe Electronicmentioning

confidence: 69%

Redox Intermediates of the Mn-Fe Site in Subunit R2 of Chlamydia trachomatis Ribonucleotide Reductase

Voevodskaya

Lendzian

Sanganas

et al. 2009

Journal of Biological Chemistry

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The R2 protein of class I ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) can contain a Mn-Fe instead of the standard Fe-Fe cofactor. Ct R2 has a redox-inert phenylalanine replacing the radical-forming tyrosine of classic RNRs, which implies a different mechanism of O 2 activation. We studied the Mn-Fe site by x-ray absorption spectroscopy (XAS) and EPR. Reduced R2 in the R1R2 complex (R2 red ) showed an isotropic six-line EPR signal at g ϳ 2 of the Mn ( Ribonucleotide reductases (RNRs) 3 are the only enzymes that, in all organisms, catalyze the reduction of ribonucleotides to their deoxy forms essential for DNA synthesis (1-3). RNRs also are important targets in cancer and antiviral therapy (4, 5).Class I RNRs found in eukaryotes and microorganisms (6) are heterotetrameric enzymes of R1 2 R2 2 organization. The R1 protein contains the nucleotide binding site and R2 houses a dinuclear metal center, which is the site of dioxygen (O 2 ) activation and, in conventional RNRs, is of the Fe-Fe type (7).Extensive investigations on Fe-Fe RNRs from, e.g. Escherichia coli, Saccharomyces cerevisiae, Mus musculus, and Homo sapiens have established that the catalytic reactions involve activation of an O 2 molecule at the di-metal cluster to generate a high potential site, which oxidizes a nearby tyrosine residue to a tyrosyl radical, Y ⅐ (8 -10). In E. coli R2 this Tyr-122 is at ϳ6 Å distance to the nearest iron (11, 12). Subsequent proton-coupled electron transfer (13) leads to the re-reduction of Y ⅐ and to the oxidation of a cysteine at the substrate binding site in R1 to a radical (C ⅐ ) (14, 15). C ⅐ initiates ribonucleotide reduction involving disulfide formation by two additional cysteines (16). Regeneration of reduced cysteines requires electron input from external thio-or glutaredoxins and ultimately from NADPH (17).At least the Fe(II) 2 , Fe(III) 2 , Fe(IV)Fe(III), and Fe(IV) 2 oxidation states of the metal center seem to be involved in the electron transfer reactions (18, 19) of classic RNRs. The Fe(III)-Fe(IV) state, termed "intermediate X" (20,21), is crucial because it oxidizes the tyrosine to Y ⅐ , leaving the di-iron site in the Fe(III) 2 state. Y ⅐ usually survives a large number of catalytic cycles, but when it is lost, the inactive Fe(III) 2 -Met form remains (22). Reactivation of the enzyme first requires reduction of the metal site to Fe(II) 2 , which then must react with O 2 , leading to the cleavage of the O-O bond and again to the formation of Fe(III) 2 and Y ⅐ (22, 23).According to the above reaction sequences, a tyrosine radical and the Fe(IV)Fe(III) state (X) have been anticipated to be decisive for RNR function. This view has been challenged recently because R2 proteins of RNRs in several species have been discovered (24, 25), containing a redox-inert phenylalanine instead of the tyrosine. One enzyme is found in the important human pathogenic bacterium Chlamydia trachomatis (Ct) (25,26). It is a fully functional RNR and the only RNR encoded in the genome of this organism (27,28). ...

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“…1). Alignments show that the Rv0233 group does not contain the conserved C-terminal tyrosine, known to participate in radical transfer from R2 to the catalytic R1 subunit and shown to be essential for activity in CtR2c (9,22) (Fig. S1).…”

Section: R2c-like Proteins Form 2 Subgroupsmentioning

confidence: 99%

A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold

Andersson

Högbom

2009

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

138

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Chlamydia trachomatis R2c is the prototype for a recently discovered group of ribonucleotide reductase R2 proteins that use a heterodinuclear Mn/Fe redox cofactor for radical generation and storage. Here, we show that the Mycobacterium tuberculosis protein Rv0233, an R2 homologue and a potential virulence factor, contains the heterodinuclear manganese/iron-carboxylate cofactor but displays a drastic remodeling of the R2 protein scaffold into a ligand-binding oxidase. The first structural characterization of the heterodinuclear cofactor shows that the site is highly specific for manganese and iron in their respective positions despite a symmetric arrangement of coordinating residues. In this protein scaffold, the Mn/Fe cofactor supports potent 2-electron oxidations as revealed by an unprecedented tyrosine-valine crosslink in the active site. This wolf in sheep's clothing defines a distinct functional group among R2 homologues and may represent a structural and functional counterpart of the evolutionary ancestor of R2s and bacterial multicomponent monooxygenases.bioinorganic chemistry ͉ diiron ͉ manganese ͉ monooxygenase ͉ R2c

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Branched Activation- and Catalysis-Specific Pathways for Electron Relay to the Manganese/Iron Cofactor in Ribonucleotide Reductase from Chlamydia trachomatis

Cited by 46 publications

References 57 publications

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

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

Redox Intermediates of the Mn-Fe Site in Subunit R2 of Chlamydia trachomatis Ribonucleotide Reductase

A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold

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