Ribonucleotide reductase resonance Raman

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... 

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. 

Fig. 9. Resonance Raman spectrum of tyrosine radical in purified E. coli ribonucleotide reductase (A) Pure B2 subunit (B) holoenzyme (C) metB2 subunit (lacking tyrosine radical). Note the correlation of the 1498 cm-1 band with the presence of the tyrosine radical. Reprinted with permission from Backes, G., Sahlin, M., Sjoberg, B.-M., Loehr, T.M. and Sanders-Loehr, J. (1989) Biochemistry 28, 1923-1929. Copyright 1989, American Chemical Society.

Figure 11. Resonance Raman spectrum of [Fe20(XDK)(BIDPhE)2-(N03)2 CH2Cl2 (A) dissolved in CH2Cl2 and wt ribonucleotide reductase R2 protein from E. coli, strain N6405/PSPS2, (B) dissolved in Tris buffer, pH 7.6, 5% glycerol. All spectra were recorded at room temperature with Kr ion laser excitation at 406.7 nm.

During the ferroxidation reaction, a blue color with an absorption maximum of 650 run appears. This persists in oxygen-limited conditions and decays as iron oxidation proceeds. " In frog H-chain ferritin, resonance Raman studies indicate a similar absorption is associated with an Fe(III)-tyrosinate. Harrison and Treffty have considered these and other studies and attribute the transient color to formation of a /x-l,2-peroxodiferric intermediate, which decays to a more stable /x-oxodiferric species as occurs in methane monooxygenase, ribonucleotide reductase, and model compounds. Protein radicals distinct from reactive oxygen species have been observed that have been attributed to damage caused by Fenton chemistry. ... 

Figure 7. Resonance Raman spectra of [Fe2O(XDK)(BIDPhE)2(N03)2] and the R2 subunit of ribonucleotide reductase.

A combination of spectroscopies including Mossbauer and resonance Raman were used to identify the presence of a diiron-oxo cluster with properties similar to those identified in ribonucleotide reductase (RB2) and methane monooxygenase (MMO). These enzymes all share the ability to break unactivated carbon-hydrogen bonds with a nonheme diiron cluster cofactor. Fatty acid desaturation and methane oxidation require a two-electron reduction of the diiron cluster to initiate the oxygen activation reaction. Identification of a diiron cluster in the desaturase allowed us to propose a consensus diiron-oxo binding motif consisting of two repeats of (D/E)EXXH. 

Sjoberg B-M, Sanders-Loehr J, Loehr TM. 1987. Identification of a hydroxide ligand at the iron center of ribonucleotide reductase by resonance raman spectroscopy. Biochemistry 26 4242 247. 

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