Ribonucleotide reductase spectroscopy

Bar, G., M. Bennati et al. (2001). High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy The tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast. J. Am. Chem. Soc. 123 3569-3576. 

M. Bennati, A. Weber, J. Antonie, D.L. Perlstein, J. Robblee and J. Stubbe, Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl radicals in the R2 subunit of the E. coli ribonucleotide reductase, J. Am. Chem. Soc., 2003, 125, 14988. 

One of the most powerful spectroscopic techniques for the detection and characterization of persistent and transient phenoxyls is time-resolved resonance Raman (RR) spectroscopy. Vibrational frequencies and the relative intensities of the resonance-enhanced bands have proven to be sensitive markers for tyrosyl radicals in proteins. For example, Sanders-Loehr and co-workers (31) detected the tyrosyl radical in native ribonucleotide reductase from Escherichia coli by a resonance-enhanced Raman mode at 1498 cm 1 that they assigned to the Ula Wilson mode of the tyrosyl, which is predominantly the u(C=0) stretching mode. 

For hemerythrin and ribonucleotide reductase, resonance Raman and EXAFS spectroscopy provide clear evidence for an oxo bridge (31-33,43,45, 62,63,65-67). Surprisingly, such evidence is lacking for the oxidized acid phosphatases. The VFe-o-Fe feature expected at ca. 500 cm has not been observed... 

Denysenkov, V. P., Prisner, T. F., Stubbe, J., and Bennati, M. (2006). High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 103, 13386-13390. 

Atkin, C. L., Thelander, L., Reichard, P., and Lang, G., 1973, Iron and free radical in ribonucleotide reductase. Exchange of iron and M sshauer spectroscopy of the protein B2 subunit of the Escherichia coli enzyme. J. Biol. Chem. 248 7464n7472. 

Bollinger, J. M., Tong, W. H., Ravi, N., Huynh, B. H., Edmondson, D. E., and Stuhhe, J., 1994h, Mechanism of assembly of the tyrosyl radical-diiron(III) cofactor of E. coli ribonucleotide reductase III. Kinetics of the limiting Fe reaction by optical, EPR, and M ssbauer spectroscopies. /. Am. Chem. Soc. 116 8024n8032. 

Lendzian, F., Sahlin, M., Macmillan, F., Bittl, R., Fiege, R., PTsch, S., Sj berg, B.-M., Gr%oslund, A., Lubitz, W., and Lassmann, G., 1996, Electronic structure of neutral tryptophan radicals in ribonucleotide reductase studied by EPR and ENDOR spectroscopy. J. Am. Chem. Soc. 118 8111ii8120. 

It was suggested, based on UV-visible and EPR spectroscopy, that the ribonucleotide reductase from Corynebac-terium ammoniagenes contains Mn rather than Fe in its native form. However, more recent crystallographic studies have shown that this protein is structurally identical to the native iron proteins and apparently does not contain a Mn active... 

Lukoyanov D, Barney BM, Dean DR, Seefeldt LC, Hoffman BM. Coimecting nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol and identification of the N2 binding state. Proc. Natl. Acad. Sci. U.S.A. 2007 104 1451-1455. Denysenkov VP, Prisner TF, Stubbe J, Bennati M. High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase. Proc. Natl. Acad. Sci. U.S.A. 2006 103 13386-13390. Schiemann O, Prisner TF. Long-range distance determinations in biomacromolecules by EPR spectroscopy. Quarterly Rev. Biophys. 2007 40 1-53. 

Ribonucleotide reductase consists of two different subunits, R1 and R2, that must be associated for enzymic activity. R1 and R2 have masses of around 180 and 90kDa, respectively. 2D >H NMR spectroscopy of the R2 subunit revealed that it has a highly mobile and unstructured C-terminal extension encompassing the last 25 amino acids of the protein.57 In the presence of the R1 subunit, however, this flexibility was lost, presumably because the extension facilitates the recognition and interaction between the two subunits. In agreement with the NMR results, mutagenesis studies showed that this C-terminal extension is crucially important in R1 and R2 interaction.-58... 

Structure of the Iron Center Formation of the Iron Center and Tyrosyl Radical Spectroscopy of the Diferric Iron Center Spectroscopy of the Tyrosyl Radical Redox Properties of the Iron Center Mixed-Valent Form of the Iron Center Diferrous Form of the Iron Center Inhibitors to Iron-Containing Ribonucleotide Reductase Methane Monooxygenase A. Spectroscopy of the MMOH Cluster X-Ray Structure of MMOH... 

The proposed mechanisms for the utilization of substrates and substrate analogs by the ribonucleotide reductases. Schemes 5 and 6, predict the formation of various radical intermediates. To search for these intermediates and their direct participation in these reactions, EPR spectroscopy has been employed (60). However, these attempts have not revealed the generation of new radical signals. 

It was shown by the magnetic susceptibility measurement and Mossbauer, ESR, and UN-visible spectroscopy that TBA-I shows an antiferromagnetic coupling of the two high-spin Fe centers. For example, the 8, AEq, and J values of TBA-I, diferric complexes, oxidized methane monooxygenase (abbreviated as MMOox), and oxidized ribonucleotide reductase (abbreviated as RRox) are shown in Table 1. The 5 and AEq values for TBA-I are close to those for MMOox, 1, 2, and 4 with the symmetrical iron centers, and different from those for RRox (5 = 0.53 mms, AEqi = 1.65 mms, and 82 =... 

ACP = acyl carrier protein ACPA D = ACPA desat-urase AlkB = octane 1-monooxygenase AOX = alternative oxidase DMQ hydroxylase = 5-demethoxyquinone hydroxylase EXAFS = extended X-ray absorption fine structure spectroscopy FMN = flavin mononucleotide FprA = flavoprotein A (flavo-diiron enzyme homologue) Hr = hemerythrin MCD = magnetic circular dichroism MME hydroxylase = Mg-protophorphyrin IX monomethyl ester hydroxylase MMO = methane monooxygenase MMOH = hydroxylase component of MMO NADH = reduced nicotinamide adenine dinucleotide PAPs = purple acid phosphatases PCET = proton-coupled electron transfer, PTOX = plastid terminal oxidase R2 = ribonucleotide reductase R2 subunit Rbr = rubrerythrin RFQ = rapid freeze-quench RNR = ribonucleotide reductase ROO = rubredoxin oxygen oxidoreductase XylM = xylene monooxygenase. 

Biomimetic studies are currently focused on generating diiron complexes that can serve as structural and/or electronic models for oxidation states higher than Fe " that are proposed to partake in the catalytic cycles of diiron proteins such as methane monooxygenase and ribonucleotide reductase. Mossbauer spectroscopy has played a leading role in the eharacterization... 

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