Ribonucleotide reductase amino acid radicals

The realization of the widespread occurrence of amino acid radicals in enzyme catalysis is recent and has been documented in several reviews (52-61). Among the catalytically essential redox-active amino acids glycyl [e.g., anaerobic class III ribonucleotide reductase (62) and pyruvate formate lyase (63-65)], tryptophanyl [e.g., cytochrome peroxidase (66-68)], cysteinyl [class I and II ribonucleotide reductase (60)], tyrosyl [e.g., class I ribonucleotide reductase (69-71), photosystem II (72, 73), prostaglandin H synthase (74-78)], and modified tyrosyl [e.g., cytochrome c oxidase (79, 80), galactose oxidase (81), glyoxal oxidase (82)] are the most prevalent. The redox potentials of these protein residues are well within the realm of those achievable by biological oxidants. These redox enzymes have emerged as a distinct class of proteins of considerable interest and research activity. 

The next five transition metals iron, cobalt, nickel, copper and zinc are of undisputed importance in the living world, as we know it. The multiple roles that iron can play will be presented in more detail later in Chapter 13, but we can already point out that, with very few exceptions, iron is essential for almost all living organisms, most probably because of its role in forming the amino acid radicals required for the conversion of ribonucleotides to deoxyribonucleotides in the Fe-dependent ribonucleotide reductases. In those organisms, such as Lactobacilli6, which do not have access to iron, their ribonucleotide reductases use a cobalt-based cofactor, related to vitamin B12. Cobalt is also used in a number of other enzymes, some of which catalyse complex isomerization reactions. Like cobalt, nickel appears to be much more extensively utilized by anaerobic bacteria, in reactions involving chemicals such as CH4, CO and H2, the metabolism of which was important... 

Type A PCET reactions describe amino acid radical generation steps in many enzymes, since the electron and proton transfer from the same site as a hydrogen atom [188]. Similarly, substrate activation at C-H bonds typically occurs via a Type A configuration at oxidized cofactors such as those in lipoxygenase [47, 48] galactose oxidase [189-191] and ribonucleotide reductase (Y oxidation at the di-iron cofactor, vide infra) [192]. Here, the HATs are more akin to the transition metal mediated reactions of Section 17.3.1 since the final site of the electron and proton are on site differentiated at Ae (redox cofactor) and Ap (a ligand). 

Application of EPR in the study of amino acid radicals in ribonucleotide reductase. Advanced EPR methods (high-field and high-frequency EPR, ENDOR, and ELDOR) have been employed extensively in the study of intermediate amino... 

Lendzian F. 2005. Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy. Biochim Biophys Acta 1707 67-90. 

Lactobacillus leichmanii ribonucleotide reductase has a molecular weight of 76 000, with a single polypeptide chain of about 690 amino acids. The large size of the apoenzyme probably reflects the need for it to have sites to interact with the coenzyme, a dithiol, a substrate and allosteric effectors. A transient radical species was observed during catalysis. 

However, recently it has proved possible to positively identify tryptophan radicals in cytochromec peroxidase[147] and tyrosine radicals in ribonucleotide reductase, prostaglandin H synthase and photosystem II of chloroplasts [148], This has been achieved by a combination of the techniques discussed already, but with the powerful, additional non-invasive tool of isotopic substitution. As deuterons (5=1) give different splitting than protons (S = 1/2), substituting different labelled amino-acid residues into the enzyme should reveal the nature of the radical-containing residue. This is easily achieved in an auxotrophic mutant that requires this amino acid to be supplied in the medium. The specific residue can then be identified by site-directed mutagenesis of the evolutionary conserved amino-acid residues [108,149-151]. 

Seyedsayamdost, M. R., Yee, C. S., Reece, S. Y., et al. (2006) pH rate profiles of FnY356-R2s (n = 2, 3,4) in Escherichia coli ribonucleotide reductase Evidence that Y-356 is a redox-active amino acid along the radical propagation pathway. Journal of the American Chemical Society, 128(5), 1562-1568. 

Recent EPR studies have shown that the amino acid tyrosine participates in a number of biological electron transfer reactions, including the oxidation of water to Oj in plant photosystem II, the reduction of Oj to water in cytochrome c oxidase, and the reduction of ribonucleotides to deoxyribonucleotides catalyzed by the enzyme ribonucleotide reductase. During the course of these electron transfer reactions, a tyrosine radical forms (4). The center of the EPR spectrum of the tyrosine radical in cytochrome c oxidase of the bacterium P. denitrificans occurs at 344.50 mT in a spectrometer operating at 9.6699 GHz (radiation belonging to the X band of the microwave region). Its -value is therefore... 

Lippard et al. proposed several possible mechanisms of the alkane hydroxylation [9]. One possible mechanism is shown in Scheme 3, in which an T 2,T 2-peroxo-bridged diiron(III) acts as an active intermediate which directly transfers oxygen to an alkane substrate [9]. This mechanism suggests the participation of a mercapto radical of Cys-151. This amino acid occupies the equivalent region of space to the tyrosyl radical of ribonucleotide reductase, as indicated by sequence homology and the X-ray crystallographic results [53, 55]. 

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