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The incubation of apo-[Y.sub.122]N[O.sub.2]Y-[beta]2 with [Fe.sup.2+] and [O.sub.2] has generated a diferric-N[O.sub.2][Y.sup..] that has a half-life of 40 s at 25 [degree] C. The studies have unmasked the conformational gating and they have disclosed that the N[O.sub.2][Y.sup..] is reduced to the nitrotyrosinate with biphasic kinetics.

Escherichia coli ribonucleotide reductase has catalyzed the reduction of nucleoside 5' -diphosphates into 2E-deoxynucleotides and is composed of two subunits ([alpha]2 and [beta]2). The radical propagation step is described to use specific redox-active tyrosines and to involve proton-coupled electron transfer (PCET) and 3-nitrotyrosine is incorporated in place of redox-active tyrosines to probe how the protein environment has perturbed each p[K.sub.a] in the presence of the second subunit, substrate, and allosteric effector.

Studies of two pathways associated with the inactivation of Ribonucleotide reductase (RNR) from Lactobacillus leichmannii that requires adenosylcobalamin (AdoCbl) as a cofactor are reported. The relationship identified between the nucleotide-based radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

One equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate ([F.sun.2]CTP) was used to rapidly inactivate ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor. The study results allowed to propose a mechanism of inactivation of RNR by [F.sun.2]CTP involving multiple reaction pathways which share many common features with inactivation of the class I RNRs.

ApoNrdF is loaded with [Mn.sup.II] and incubated with fully reduced NrdI ([NrdI.sub.hq]) and [O.sub.2] to explore the function of operon NrdI of homodimeric subunit found in Escherichia coli class Ib ribonucleotide reductase (RNR) and identify the metallocofactor that initiates nucleotide reduction. The results suggested that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the [Mn.sup.III.sub.2]-Y* cofactor, not the diferric-Y* one, is the active metallocofactor in vivo.

Stopped-flow (SF) UV-vis and EPR spectroscopies were used to examine the site-specific incorporation of 3-Aminotyrosine (N[H.sub.2]Y) at residues 730 and 731 and formation of the aminotyrosyl radical (N[H.sub.2]Y*). The results provide insight into the evolution of an optimized pathway of conserved diferric tyrosyl radical Ys cofactor in the oxidation of [C.sub.439].

[Alpha] with specific activity of 400 nmol [min.sup.-1] [mg.sup.-1]) and [beta]' (0.6 Y*/[beta]'2 and a specific activity of 420 nmol [min.sup.-1] [mg.sup.-1]) is used to establish [F.sub.2]CDP as a substoichiometric inactivator of RNR. The results from the analysis of the inactivated wt-[alpha]/[beta]' RNR by size exclusion chromatography indicated a quaternary structure of [alpha]6[beta]'6. which is a mechanism of inactivation common with h[alpha]/[beta].

A detailed study was conducted to gain insight into enzyme inactivation mechanism of the Escherichia coli ribonucleotide reductase (RNR), an [alpha]2[beta]2 complex incubated with 1 equiv of [F.sub.2]CDP which catalyzes the release of two fluorides and cytosine. The enzyme inactivation was found to result from its covalent labeling of [alpha] with the sugar of [F.sub.2]CDP (one label/[alpha]2[beta]2), in the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT).

Escherichia coli ribonucleotide reductase (RNR) has catalyzed the conversion of nucleotides to deoxynucleotides, a process that needs long-range radical transfer from a tyrosyl radical within the [beta]2 subunit to a cysteine residue ([C.sub.439]) within the [alpha]2 subunit. The hyperfine coupling constants and geometry of the N[H.sub.2] moiety are shown to be consistent with an intramolecular hydrogen bond within N[H.sub.2][Y.sub.730] radical.

Two mechanisms for elongation are proposed, where the first has involved an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between C149 on each monomer and the second has involved C149 and covalent and noncovalent [(HB).sub.n]CoA intermediates. The [(HB).sub.n]CoA analogues have functioned as kinetically and chemically competent intermediates and the results have supported the mechanism involving covalently and noncovalently bound intermediates.

The article studies the two mechanisms for elongation that includes an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between C149 on each monomer and includes C149 and covalent and noncovalent [(HB).sub.n]CoA intermediates. Findings support the mechanism connecting covalently and noncovalently bound intermediates.

Photochemical ribonucleotide reductases (photoRNRs) were developed to study the proton-coupled electron transfer (PCET) mechanism of radical transport in Escherichia coli class I ribonucleotide reductase (RNR). The biexponential kinetics observed for PCET suggested a large degree of conformational flexibility in the [Re]-Y-[Re]-Y-[beta]C19- [alpha]2 complex that engendered partitioning of the N-terminus of the peptide into both bound and solvent-exposed fractions.

The improvements in the 35 GHz pulsed ENDOR performance is used for reexamining the protonation state of oxygenic ligands of the inorganic core of ribonucleotide reductase intermediate X by directly probing the exchangeable protons with [super 2]H pulsed electron-nuclear double resonance (ENDOR) spectroscopy. The studies have shown that X contains a terminal aqua ligand to [Fe.sup.III] in addition to one or two [mu]-oxo bridges but does not contain a [mu]-hydroxo bridge.

A new method was developed to determine the molecular basis for interactions between the subunits of ribonucleotide reductase (RNR) and to measure their affinities in the presence of different substrate and effector pairs. The developed method could also be applied to RNRs from other sources and gain insight into the complexities of other flexible proteins.

Two forms of formyl glycinamide ribonucleotide (FGAR) amidotransferase (PurL), large and small, are characterized. The flexibility of the PurS dimer has played a major role in the activation of the complex and the formation of the ammonia channel.

The role of diferric tyrosyl radical ([Y.sup.*]) cofactor in the regulation of ribonucleotide reductases (RNR) activity in Escherichia coli is investigated. N- and C-terminal StrepII-tagged [beta] under the control of an L-arabinose promoter are constructed for use in the study.

The studies of site-specific integration of 3-aminotyrosine (N[H.sub.2]Y) into one of the subunits of Escherichia coli ribonculeotide reductase (RNR) is reported. The findings provide insight into role of [Y.sub.730] and [Y.sub.731] mutants related to the mechanism of radical initiation.

The proton-coupled electron transfer (PCET) of radical transport in ribonucleotide reductase (RNR) is studied by using engineered photoRNRs. The [Re] chromophore with fluorotyrosines has provided a method for selectively generating tyrosyl radicals on the [F.sub.R]Y-R2C19 peptide bound to [alpha]2 that are competent for turnover and that can be seen with transient absorption (TA) spectroscopy.

The article discusses the discovery of a 2Fe2S ferredoxin, YfaE in Escherichia coli. Evidences reveal that it plays an important role in diferric-tyrosyl radical cofactor maintenance.

The phototriggered generation of a light-initiated tyrosyl radical is found to be extremely helpful for the study of the mechanism of radical transport in ribonucleotide reductase (RNR). The analysis reveals that the radical transport is actually a unidirectional transport of the electron and proton among the residues.

The structure of 4'-oxidized abasic site (X) is determined by two-dimensional nuclear magnetic resonance (NMR) methods, molecular modeling and molecular dynamics simulations. Molecular dynamics simulations have shown that the normal abasic site is more flexible than the 4'-oxidized abasic site.

The different structures of formylglycinamide ribonucleotide amidotransferase (FGAR-AT) from Thermotoga maritima are determined in the presence of substrates, a substrate analogue and a product, thereby allowing a detailed description of the novel ATP-binding motif. An auxiliary site of TmPurL, structurally equivalent to the auxiliary ADP binding site of StPurL, is found to bind an ATP molecule and also play regulatory role in the formation of the PurLSQ complex.

A study demonstrates that a tyrosyl radical is generated promptly upon photoexcitation, and it is of sufficient lifetime to promote the investigation of radical transport. One reason for the prompt photoregeneration is that the non-natural [F.sub.n]Y amino acids are deprotonated at physiological pHs so that radical initiation occurs by simple electron transfer.

The in vivo stoichiometry of tyrosyl radical (Y*) cofactor per [beta][beta'] R2 subunit in Saccharomyces cerevisiae ribonucleotide reductase (RNR) is determined by whole-cell EPR studies and activity measurements of crude cell extracts. Results suggest that in asynchronously growing cells, RNR activity is not modulated at the level of active Y* cofactor formation, and that the subunits are at concentrations much greater than those required for genome replication suggesting that their activities must be greatly modulated to become rate-limiting for DNA replication.

Experiments that provide further support for the hypothesis that the heterodimer (betabeta') is the active form of yeast R2 in vitro and to further examine the active form of R2 in vivo is presented. Results demonstrate that Beta' plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is betabeta'.

The first direct evidence for (HB)n-Allochromatium vinosum [PhaC(sub AV)] intermediates generated with the class III synthase and HB-CoA is reported. Results reveal that at low S/E ratios, species I was observed to increase with time after complete consumption of all of the hydroxybutyrate-coenzyme A(HB-CoA).

The incubation of D302A-PhaCPhaE with [(super 14)C]-HB-CoA results in detection of oligomeric HBs covalently bound to PhaC is reported. The in vitro results support the role of D302 in elongation rather than in activating the active site cysteine acylation.

Radiolytic reduction of diferric desaturase (DS) at 77K produces an electron paramagnetic resonance (EPR)-detectable mixed-valence center that is trapped in the conformation of the deferric precursor and thus provides a sensitive EPR/electron nuclear double resonance (ENDOR) probe of the structure of the diamagnetic diiron(III) state. The cyroreduced DS shows two distinct EPR signals, suggesting the presence of two diiron(III) states, the Mu-oxo- and Mu-hydroxo-bridged diiron centers.