Structural Analysis of the Mn(IV)/Fe(III) Cofactor of Chlamydia trachomatis Ribonucleotide Reductase by Extended X-ray Absorption Fine Structure Spectroscopy and Density Functional Theory Calculations

Younker, Jarod M.; Krest, Courtney M.; Jiang, Wei; Krebs, Carsten; Bollinger, J. Martin; Green, Michael T.

doi:10.1021/ja804365e

Cited by 55 publications

(131 citation statements)

References 57 publications

(131 reference statements)

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“…CtR2 MnFe red showed opposite magnitudes (ϳ25%, ϳ50%) of contributions from these two distances. Similar metal-metal distances in CtR2 MnFe have previously been attributed to Mn(IV)/(III)Fe(III) (ϳ2.90 Å) and Mn(II)Fe(III) (ϳ3.3 Å) sites (29,30,66). Here, the ϳ2.90 Å distance mostly reflected the Mn(III)Fe(III) cofactor, because Mn(IV) contributions were small.…”

Section: Structure and Xpr Of The Mnfe Cofactor-the Xpr Kinetics Of Ctr2supporting

confidence: 79%

“…Implications for X-ray Crystallography of Dimetal-Oxygen Cofactors-Our present and previous data (29,30,66) indicate that XPR of high-valent FeFe and MnFe sites in CtR2 and also in the standard type R2 protein from mouse can occur within seconds to minutes already under XAS conditions. The respective doses (at 80 K) are at least 100 times smaller than those resulting in radiation damage of amino acid groups (46,105).…”

Section: Structures Of Fefe and Mnfe Cofactors-ourmentioning

confidence: 54%

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Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

Sigfridsson

Chernev

Leidel

et al. 2013

Journal of Biological Chemistry

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Background: Typical FeFe and MnFe cofactors bind to numerous enzymes such as ribonucleotide reductases. Crystallographic data suggest x-ray photoreduction (XPR) effects. Results: Rapid XPR-induced cofactor changes were monitored using time-resolved x-ray absorption spectroscopy. Conclusion: The XPR-induced cofactor states differ significantly from the native configurations, but comply with crystallographic structures. Significance: Structure determination for high-valent dimetal-oxygen cofactors requires free electron-laser protein crystallography combined with x-ray spectroscopy.

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Section: Structure and Xpr Of The Mnfe Cofactor-the Xpr Kinetics Of Ctr2supporting

confidence: 79%

Section: Structures Of Fefe and Mnfe Cofactors-ourmentioning

confidence: 54%

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

Sigfridsson

Chernev

Leidel

et al. 2013

Journal of Biological Chemistry

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“…The velocity of x-ray photoreduction suggests a value close to 1 V (62). Interestingly, Mn(IV) is more rapidly reduced than Fe(III) (27,48). These results point to a Mn(IV) Ͼ Fe(III) Ͼ Mn(III) order of the oxidizing potentials.…”

Section: Mn-fe Electronicmentioning

confidence: 77%

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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“…95,[96][97][98][99][100][101] For recent reviews see. [102][103][104][105] Notably, it was shown that the reconstitution reaction proceeds via a Mn(IV)-Fe(IV) Intermediate state.…”

Section: Class Ic R2 Proteinsmentioning

confidence: 99%

Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear—an intricate bioinorganic workaround to use different metals for the same reaction

Högbom

2011

Metallomics

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This is an author produced version of a paper published in [ METALLOMICS ]This paper has been peer-reviewed but may not include the final publisher proof-corrections or journal pagination. Citation 2(42) AbstractThe ferritin-like superfamily comprises several protein groups that utilize dinuclear metal sites for various functions, from iron storage to challenging oxidations of substrates.Ribonucleotide reductase R2 proteins use the metal site for generation of a free radical required for the reduction of ribonucleotides to deoxyriboinucleotides, the building blocks of DNA. This ubiquitous and essential reaction has been studied for over four decades and the R2 proteins were, until recently, generally believed to employ the same cofactor and mechanism for radical generation. In this reaction, a stable tyrosyl radical is produced following activation and cleavage of molecular oxygen at a dinuclear iron site in the protein.Discoveries in the last few years have now firmly established that the radical generating reaction is not conserved among the R2 proteins but that different subgroups, that are structurally very similar, instead employ di-manganese or heterodinuclear Mn-Fe cofactors as radical generators. This is remarkable considering that the protein must exercise a strict control over oxygen activation, reactive metal-oxygen intermediate species and the resulting redox potential of the produced radical equivalent. Given the differences in redox properties between Mn and Fe, use of a different metal for this reaction requires associated adaptations of the R2 protein scaffold and the activation mechanism. Further analysis of the differences in protein sequence between R2 subgroups have also led to the discovery of new groups of R2-like proteins with completely different functions, expanding the chemical repertoire of the ferritin-like superfamily. This review describes the discoveries leading up to the identification of the different Mn-containing R2 protein groups and our current understanding of them.Hypotheses regarding the biochemical rationale to develop these chemically complex alternative solutions are also discussed.

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Structural Analysis of the Mn(IV)/Fe(III) Cofactor of Chlamydia trachomatis Ribonucleotide Reductase by Extended X-ray Absorption Fine Structure Spectroscopy and Density Functional Theory Calculations

Cited by 55 publications

References 57 publications

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

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

Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear—an intricate bioinorganic workaround to use different metals for the same reaction

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