High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(µO)(2)Fe(III)L(4) (metal-metal distance of ~2.75Å, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(µO)(µOH)Fe(III)L(4) (~2.90Å) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55Å) with a L(4)Fe(IV)(µO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1Å) and Mn,Fe(III)Fe(II) species (~3.3-3.4Å) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(µO)(µOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.

Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination.

In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.

Metal binding and activity of ribonucleotide reductase protein R2 mutants: conditions for formation of the mixed manganese-iron cofactor.

Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

Structure of the high-valent FeIIIFeIV state in ribonucleotide reductase (RNR) of Chlamydia trachomatis--combined EPR, 57Fe-, 1H-ENDOR and X-ray studies.

A recently discovered subgroup of class I ribonucleotide reductase (RNR) found in the infectious bacterium Chlamydia trachomatis (C. trachomatis) was shown to exhibit a high-valent Fe(III)Fe(IV) center instead of the tyrosyl radical observed normally in all class I RNRs. The X-ray structure showed that C. trachomatis WT RNR has a phenylalanine at the position of the active tyrosine in Escherichia coli RNR. In this paper the X-ray structure of variant F127Y is presented, where the tyrosine is restored. Using (1)H- and (57)Fe-ENDOR spectroscopy it is shown, that in WT and variants F127Y and Y129F of C. trachomatis RNR, the Fe(III)Fe(IV) center is virtually identical with the short-lived intermediate X observed during the iron oxygen reconstitution reaction in class I RNR from E. coli. The experimental data are consistent with a recent theoretical model for X, proposing two bridging oxo ligands and one terminal water ligand. A surprising extension of the lifetime of the Fe(III)Fe(IV) state in C. trachomatis from a few seconds to several hours at room temperature was observed under catalytic conditions in the presence of substrate. These findings suggest a possible new role for the Fe(III)Fe(IV) state also in other class I RNR, during the catalytic radical transfer reaction, by which the substrate turnover is started.

High catalytic activity achieved with a mixed manganese-iron site in protein R2 of Chlamydia ribonucleotide reductase.

Ribonucleotide reductase (class I) contains two components: protein R1 binds the substrate, and protein R2 normally has a diferric site and a tyrosyl free radical needed for catalysis. In Chlamydia trachomatis RNR, protein R2 functions without radical. Enzyme activity studies show that in addition to a diiron cluster, a mixed manganese-iron cluster provides the oxidation equivalent needed to initiate catalysis. An EPR signal was observed from an antiferromagnetically coupled high-spin Mn(III)-Fe(III) cluster in a catalytic reaction mixture with added inhibitor hydroxyurea. The manganese-iron cluster in protein R2 confers much higher specific activity than the diiron cluster does to the enzyme.

Redox intermediates of the Mn-Fe Site in subunit R2 of Chlamydia trachomatis ribonucleotide reductase: an X-ray absorption and EPR study.

The R2 protein of class I ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) can contain a Mn-Fe instead of the standard Fe-Fe cofactor. Ct R2 has a redox-inert phenylalanine replacing the radical-forming tyrosine of classic RNRs, which implies a different mechanism of O(2) activation. We studied the Mn-Fe site by x-ray absorption spectroscopy (XAS) and EPR. Reduced R2 in the R1R2 complex (R2(red)) showed an isotropic six-line EPR signal at g approximately 2 of the Mn(II)Fe(II) state. In oxidized R2 (R2(ox)), the Mn(III)Fe(III) state exhibited EPR g values of 2.013, 2.009, and 2.015. By XAS, Mn-Fe distances and oxidation states of intermediates were determined and assigned as follows: approximately 4.15 A, Mn(II)Fe(II); approximately 3.25 A, Mn(III)Fe(II); approximately 2.90 A, Mn(III)Fe(III); and approximately 2.75 A, Mn(IV)Fe(III). Shortening of the Mn/Fe-ligand bond lengths indicated formation of additional metal bridges, i.e. microO(H) and/or peroxidic species, upon O(2) activation at the site. The structural parameters suggest overall configurations of the Mn-Fe site similar to those of homo-metallic sites in other R2 proteins. However, the approximately 2.90 A and approximately 2.75 A Mn-Fe distances, typical for di-microO(H) metal bridging, are shorter than inter-metal distances in any R2 crystal structure. In diffraction data collection, such bridges may be lost due to rapid x-ray photoreduction of high-valent metal ions, as demonstrated here for Fe(III) by XAS.

Structural and biophysical characterization of human myo-inositol oxygenase.

Altered inositol metabolism is implicated in a number of diabetic complications. The first committed step in mammalian inositol catabolism is performed by myo-inositol oxygenase (MIOX), which catalyzes a unique four-electron dioxygen-dependent ring cleavage of myo-inositol to D-glucuronate. Here, we present the crystal structure of human MIOX in complex with myo-inosose-1 bound in a terminal mode to the MIOX diiron cluster site. Furthermore, from biochemical and biophysical results from N-terminal deletion mutagenesis we show that the N terminus is important, through coordination of a set of loops covering the active site, in shielding the active site during catalysis. EPR spectroscopy of the unliganded enzyme displays a two-component spectrum that we can relate to an open and a closed active site conformation. Furthermore, based on site-directed mutagenesis in combination with biochemical and biophysical data, we propose a novel role for Lys(127) in governing access to the diiron cluster.

The involvement of Arg265 of mouse ribonucleotide reductase R2 protein in proton transfer and catalysis.

Ribonucleotide reductase class I enzymes consist of two non-identical subunits, R1 and R2, the latter containing a diiron carboxylate center and a stable tyrosyl radical (Tyr*), both essential for catalysis. Catalysis is known to involve highly conserved amino acid residues covering a range of approximately 35 A and a concerted mechanism involving long range electron transfer, probably coupled to proton transfer. A number of residues involved in electron transfer in both the R1 and R2 proteins have been identified, but no direct model has been presented regarding the proton transfer side of the process. Arg265 is conserved in all known sequences of class Ia R2. In this study we have used site-directed mutagenesis to gain insight into the role of this residue, which lies close to the catalytically essential Asp266 and Trp103. Mutants to Arg265 included replacement by Ala, Glu, Gln, and Tyr. All mutants of Arg265 were found to have no or low catalytic activity with the exception of Arg265 to Glu, which shows approximately 40% of the activity of native R2. We also found that the Arg mutants were capable of stable tyrosyl radical generation, with similar kinetics of radical formation and R1 binding as native R2. Our results, supported by molecular modeling, strongly suggest that Arg265 is involved in the proton-coupled electron transfer pathway and may act as a proton mediator during catalysis.

Loss of the tyrosyl radical in mouse ribonucleotide reductase by (-)-epicatechin.

The flavonoid (-)-epicatechin was previously demonstrated to interfere with tyrosine nitration by peroxynitrite [Biochem. Biophys. Res. Commun. 285 (2001) 782]. This effect was hypothesized to be based upon an interaction of epicatechin with a transiently generated tyrosyl radical. In the present study, using electron paramagnetic resonance, we demonstrate that (-)-epicatechin is capable of destabilizing the tyrosyl radical of the mouse ribonucleotide reductase R2 component. First-order rate constants for the disappearance of tyrosyl radical signals were 1 x 10(-4) and 2 x 10(-4)s(-1)for epicatechin and hydroxyurea, a well-known tyrosyl radical scavenger, respectively. In keeping with scavenging the ribonucleotide reductase tyrosyl radical, cellular production of deoxyribonucleotides and DNA synthesis were impaired by (-)-epicatechin in normal human keratinocytes and in human squamous carcinoma cells.

The radical site in chlamydial ribonucleotide reductase defines a new R2 subclass.

Ribonucleotide reductase (RNR) synthesizes the deoxyribonucleotides for DNA synthesis. The R2 protein of normal class I ribonucleotide reductases contains a diiron site that produces a stable tyrosyl free radical, essential for enzymatic activity. Structural and electron paramagnetic resonance studies of R2 from Chlamydia trachomatis reveal a protein lacking a tyrosyl radical site. Instead, the protein yields an iron-coupled radical upon reconstitution. The coordinating structure of the diiron site is similar to that of diiron oxidases/monoxygenases and supports a role for this radical in the RNR mechanism. The specific ligand pattern in the C. trachomatis R2 metal site characterizes a new group of R2 proteins that so far has been found in eight organisms, three of which are human pathogens.

EPR studies of the mitochondrial alternative oxidase. Evidence for a diiron carboxylate center.

The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (-J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.

Exogenous ferrous iron is required for the nitric oxide-catalysed destruction of the iron-sulphur centre in adrenodoxin.

No effects of gaseous NO added at a pressure of 19.95 kPa on the stability of the binuclear iron-sulphur centre (ISC) of reduced iron-sulphur protein adrenodoxin (0.2 mM) have been observed using the EPR method. However, the incubation of the protein with NO in the presence of ferrous iron (1.8 mM) led to complete ISC degradation, accompanied by the formation of protein-bound dinitrosyl iron complexes (DNICs; 0.3+/-0.1 mM). Similar results were obtained when low-molecular-mass DNIC with phosphate or cysteine (1.8 mM) were added to solutions of pre-reduced adrenodoxin. The degradation of the ISC was suggested to be due to the attack of the Fe(+)(NO(+))(2) group from low-molecular-mass DNICs added or formed during the interaction between NO and ferrous ions on the thiol groups in active centres of adrenodoxin. This attack leads to a release of endogenous iron from the centres, which is capable of forming both low-molecular-mass and protein-bound DNIC, thereby ensuring further ISC degradation.