Ribonucleotide reductase R2 proteins

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.

Davydov, A., Schmidt, P. P., and Gr%oslund, A., 1996a, Reversible red-ox reactions of die diiron site in the mouse ribonucleotide reductase R2 protein. Biochem. Biophys. Res. Comimm. 219 213n218. 

Rova, U., Adrait, A., PTsch, S., Gr%oslund, A., and Thelander, L., 1999, Evidence by mutagenesis that Tyr(370) of the mouse ribonucleotide reductase R2 protein is the connecting link in the intersubunit radical transfer pathway. J. Biol. Chem. 274 23746n23751. 

Figure 4 a) X-band EPR spectra of tyrosyl free radical in (i) E. coli, (ii) Mycobacterium tuberculosis, and (iii) mouse ribonucleotide reductase R2 proteins (1 7). All spectra were obtained under nonsaturation conditions at 20 K. b) Spin density distribution of the unpaired electron obtained from Isotope-labeling EPR studies, c) The distances between the phenolic oxygen of tyrosyl radical and the nearest Fe ion deduced from the relaxation properties of the tyrosyl radicals. 

Jiang W, Bollinger JM Jr, Krehs C. The active form of Chlamydia trachomatis ribonucleotide reductase R2 protein contains a het-erodinuclear Mn(IV)/Fe(III) cluster with S = 1 ground state. J. Am. Chem. Soc. 2007 129 7504-7505. 

A number of other metalloenzymes have M 2(His)2(02CR)4 active sites (see Chapter 8.13). Most prominent of these are the di-iron enzymes, including the hydroxylase component of methane monooxygenase (MMOH, Figure 15a) and class 1 ribonucleotide reductase R2 proteins (Figure ISb). " These enzymes have two conserved Asp/Glu(Xaa)nGluXaaXaaHis sequence motifs in a four-helix bundle that provide the six amino-acid ligands for the di-iron... 

Figure 13.25 Three-dimensional structures of diiron proteins. The iron-binding subunits of (a) haemery-thrin, (b) bacterioferritin, (c) rubryerythrin (the FeS centre is on the top), (d) ribonucleotide reductase R2 subunit, (e) stearoyl-acyl carrier protein A9 desaturase, (f) methane monooxygenase hydroxylase a-subunit. (From Nordlund and Eklund, 1995. Copyright 1995, with permission from Elsevier.)...

Fig. 1. Diferric iron clusters form hemer3fthrin, ribonucleotide reductase R2 subunit, and methane monooxygenase hydroxylase. The figure was made with the RasMol 2.0 program, and the protein coordinates as PDB files were obtained from Brookhaven Protein Data Bank. Only the amino acids (histidines, green carboxylates, black oxygen, red nitrogen, yellow acetate, blue iron, violet) coordinated to the iron cluster are shown, coordinated waters are not indicated. The first subunit containing the cluster is shown. Diferric Hr is from sipunculid worm Themiste dyscritra). The RNR-R2 is from E. coli. The MMOH is from Methvlococcus caosulatus (Bath).

Although they show little overall sequence identity, the dinuclear centers of the three prototype ferritins, particularly that of EcBfr, are remarkably similar in both amino acid residues and their geometry to those of the class 2 diiron proteins that comprises the ribonucleotide reductase R2 subunit (RNR R2), the methane monooxygenase hydroxylase component (MMOH), rubrerythrin (Rr) and several other... 

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. 

Rubrerythrin (Rr) was first isolated in 1988 from cellular extracts of D. vulgaris Hildenborough (38), and later also found in D. desulfuri-cans (39). Rr is constituted by two identical subunits of 22 kDa and it was shown that each monomer contains one Rd-like center, Fe(RS)4, and a diiron-oxo center similar to the ones found in methane monooxygenase (MMO) (40, 41) or ribonucleotide reductase (RNR-R2) (42). After aerobic purification, the UV-visible spectrum shows maxima at 492, 365, and 280 nm, and shoulders at 570 and 350 nm. This spectrum is similar to the ones observed for Rd proteins. From a simple subtraction of a typical Rd UV-vis spectrum (normalized to 492 nm) it is possible to show that the remainder of the spectrum (maxima at 365 nm and a shoulder at 460 nm) strongly resembles the spectrum of met-hemerythrin, another diiron-oxo containing protein. 

Davydov, R., Kuprin, S. Graslund, A., and Ehrenberg, A. 1994. Electron paramagnetic resonance study of the mixed-valent diiron center in Escherichia coli ribonucleotide reductase produced by reduction of radical-free protein R2 at 77 K. Journal of the American Chemical Society 116 11120-11128. 

Figure 5. Active site structure of the met form of the E. coli R2 protein of ribonucleotide reductase as determined in a 2.2-A resolution X-ray crystallographic study (14, 102).

Figure 9. A low-molecular weight model complex for the met form of the R2 protein of ribonucleotide reductase. [After (136, 137).]...

Dinuclear iron centres occur in several proteins. They either bind or activate dioxygen or they are hydrolases. Ribonucleotide reductase (RR) of the so-called class I type contains one such centre in the R2 protein in combination with a tyrosyl radical, both being essential for enzymatic activity which takes place in the R1 protein subunit. The diiron centre activates dioxygen to generate the tyrosyl radicals which in turn initiate the catalytic reaction in the R1 subunit. The interplay between the tyrosyl free radical in R2 and the formation of deoxyribonucleotides in R1 which also is proposed to involve a protein backbone radical is a topic of lively interest at present but is outside the scope of this review. Only a few recent references dealing with this aspect are mentioned without any further discussion.158 159 1 1,161... 

In subunit R2 of ribonucleotide reductase there is a tyrosyl radical (Y ) in close proximity to a di-iron cluster.100 In the protein from E. coli the EPR signal from Y can be observed up to room temperature. However, in the protein from yeast the Y signal broadens above 15 K and is not observable above about 60 K. Saturation recovery measurements at 140 GHz showed that at 60 K the spin-lattice relaxation rates for the Y signal in the yeast protein were about 2 orders of magnitude faster than for the E. coli protein. The temperature dependence of the relaxation enhancement was consistent with the activation energy for the first excited state of the di-iron cluster, so the relaxation enhancement was attributed to interaction with the di-iron cluster. Relaxation enhancements measured at 140 GHz showed little orientation dependence so the enhancement was assigned to isotropic exchange, which is different from the orientation-dependent dipolar interaction observed for the E. coli protein.100... 

Figure 1. Structure of the R2 protein of E. coli ribonucleotide reductase in the met form. (Adapted from reference 7, which reports the 2.2 A crystal structure of the protein. Note that, in reference 7, Asp 84 is considered to be bidentate and chelating, but we prefer the mono dentate, hydrogen-bonded representation depicted above based on an analysis of the Fe-O distances.)...

Figure 2. Structure of a S211A mutant of the R2 protein of E. coli ribonucleotide reductase in the reduced form. (Adapted from reference 11 reporting the 2.2 A crystal structure.)...

Nordlund, P., and Eklund, H., 1993, Structure and function of the Escherichia coli ribonucleotide reductase protein R2 J. Mol. Biol. 232 1239164. 

Bollinger, J. M., Krehs, C., Vicol, A., Chen, S. X., Ley, B. A., Edmondson, D. E., and Huynh, B. H., 1998, Engineering the diiron site of Escherichia coli ribonucleotide reductase protein R2 to accumulate an intermediate similar to H-peroxo, the putative peroxodiiron(III) complex from the methane monooxygenase catalytic cycle. J. Am. Chem. Soc. 120 1094nl095. 

Climent, L, Sj berg, B.-M., and Huang, C. Y., 1992, Site-directed mutagenesis and deletion of the carboxyl terminus of Escherichia coli ribonucleotide reductase protein R2. Effects on catalytic activity and subunit interaction. Biochemistry 31 4801fi4807. 

Logan, D. T, deMarE, F., Persson, B. O., Slaby, A., Sj berg, B.-M., and Nordlund, P., 1998, Crystal structures of two self-hydroxylating ribonucleotide reductase protein R2 mutants Structural basis for the oxygen-insertion step of hydroxylation reactions catalyzed by diiron proteins. Biochemistry 37 10798nl0807. 

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