Ribonucleotide Reductase RR

The crystal structure of the mouse R2 RR has also been solved but it contained only one iron ion, maybe because in this enzyme the iron/radical site is more exposed to the solvent [72]. A recent report on the crystal structure of this mamma-Han R2 with a dinuclear cobalt center replacing iron foimd a different orientation for one of the glutamate Hgands when compared with the bacterial enzyme [73]. This could explain the increased labiHty of one of the iron centers. Because the substrate-binding site in R1 and the radical-generating site in R2 are far apart a 

In class II RRs, which are homodimeric enzymes, the radical species is generated by adenosyl cobalamine. They can fimction either aerobically or anaerobically and they are not found in higher organisms [78]. Anaerobic class III RRs have a a2 2 quaternary structure, they contain an FeS cluster and a glycyl radical and belong to the radical enzyme family described below. No structure is available for class III RRs [79]. 

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A new class of metalloprotelns containing polynuclear, non-heme oxo-bridged iron complexes has emerged recently. Dinuclear centers are present in hemerythrin (Hr), ribonucleotide reductase (RR), purple acid phosphatases (PAP) and, possibly, methane monooxygenase (MMO) these centers as well as model compounds are reviewed in Chapter 8. 

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... 

The critical and rate-controlling step in the pathway leading to the synthesis of DNA is focused on ribonucleotide reductase (RR). Inhibitors of RR would have great utility as a therapeutic agent against cancer. One such potent inhibitor of ribonuclease diphosphate reductase is 3-AP 196, and a Suzuki methylation reaction that converted 194 into 195 began the synthesis of this compound [69]. 

A connection between the manganese catalyzed formation of benzyl radicals and tyrosyl radicals can be formally established. It is known that Mn can replace Fe in some ribonucleotide reductases (RR) with retention of activity. Also, a dinuclear Mn active site has been proposed for authentic manganese ribonucleotide reductases (MnRR).l Both RR s share the X-oxo bridged dinuclear manganese motif with 1, except that, as discussed above, 1 comprises two such structural units. 

Sequence Comparison Between the N-Terminal Part of the Fepr Genes from Desulfovibrio desulfuricans (Dd) and Desulfovibrio vulgaris (Dv), Carbon Monoxide Dehydrogenase from Methanothrix soehngenii (Ms), Methanosarcina frisia Gdl (Mf), Clostridium thermoaceticum (Ct), Rhodospirillum rubrum (Rr), and Anaerobic Ribonucleotide Reductase from Escherichia coli (Ec) ... 

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. 

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. 

The presentation in 1993 of the structure of the hydroxylase component of methane monooxygenase (MMOH) by Rosenzweig et al. (15) is the third published three-dimensional structure of a diiron-oxygen protein (Fig. 1). The previous two are from hemerythrin (Hr) (16,17) and protein R2 of E. coli ribonucleotide reductase (RNR-R2) (18, 19). Some other dinuclear iron proteins with known fi-oxo or p.-hydroxo bridges are purple acid phosphatases (PAP) [(e.g., uteroferrin (Uf)] (20, 21), ferritins (in early stages of nucleation) (22), rubrerythrin (Rr) (23-26), nigerythrin (26), and soluble stearoyl-acyl carrier protein A desaturase (A-AGP) (27, 28). 

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... 

Fig. 41.2. Overview of purine production, starting with glutamine, ribose 5-phosphate, and ATP. The steps that require ATP are also indicated in this figure. RR = ribonucleotide reductase. FH4 = tetrahydrofolate. PRPP = 5-phosphoribosyl 1-pyrophosphate.

Fig. 41.14. Synthesis of the pyrimidine bases. CPSn = carbamoyl phosphate synthetase II. RR = ribonucleotide reductase = stimulated by 0 = inhibited by FH2 and FH4 = forms of folate.

A protein may have more than one metal center, each with its own set of electronic transitions, and in favorable cases the RR spectra can be elicited selectively by judicious choice of laser wavelengths. For example, sulfite reductase has an iron-isobacteriochlorin cofactor, siroheme, which is coupled magnetically to a Fe4S4 cluster, probably through a thiolate bridge. The siroheme modes are strongly enhanced in resonance with the isobacteriochlorin Soret or Q bands, but intermediate excitation (457.9-488.0 nm) enhances the Fe4 4 cluster modes (Fig. 11). The protein rubrerythrin has two rubredoxin-like Fe(Cys)4 centers and a (/u.-oxo)di-iron(III) center (similar to those found in hemerythrins and ribonucleotide reductase), each of which can be enhanced selectively by excitation at... 

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