A New Tyrosyl Radical on Phe208 as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H

Abstract

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr 122 ), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe 208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.Over the past decades a number of structurally related proteins that contain oxygen-bridged dinuclear iron centers have been discovered and characterized (1, 2). Among these are the hydroxylase protein component of methane monooxygenase (MMO), 1 called MMOH (3-5), and the R2 protein component of class I ribonucleotide reductase (RNR) (6 -8). These proteins reveal a strikingly similar coordination arrangement of their diiron centers, but fulfill entirely different biological functions; in MMOH, the diiron center is located directly at the substrate binding catalytic site and is directly responsible for the hydroxylation of methane or other substrates, whereas in R2, the diiron center is more than 35 Å away from the substrate binding active site located in the second protein subunit R1 (9 -11). The catalytic role of the diiron center in R2 is to generate and stabilize a tyrosyl radical (at position Tyr 122 in Escherichia coli R2), and this tyrosyl radical is connected to the active site in R1 via a chain of H-bonded amino acid residues. It has been proposed that upon substrate binding in R1 Tyr 122 transfers its radical character via this chain to a cysteine (Cys 439 in E. coli R1), which in turn starts substrate turnover from ribonucleotide to deoxyribonucleotide (9 -12).The R2 protein is a homodimer which in its active form contains two -oxo bridged diferric iron centers and a substoichiometric amount of a tyrosyl radical. The active form can be generated in vitro by the so-called reconstitution reaction by adding a 6-fold molar excess of Fe 2ϩ and molecular oxygen to the iron-free apoR2 protein (13). The tyrosyl radical is stabilized by a surrounding cluster of hydrophobic side ch...

Epoxy Coenzyme A Thioester Pathways for Degradation of Aromatic Compounds

“…BoxB is a diiron enzyme instead of a protein containing a Rieske [2Fe-2S] cluster and a mononuclear iron. Although BoxB is a class I diiron enzyme, it is still quite distinct from known examples such as stearyl-ACP ⌬9 desaturase, ribonucleotide reductase, soluble methane monooxygenase, or the above-described phenylacetyl-CoA epoxidase (27,28,49,53,54,55). It is monomeric and not part of a multimeric enzyme complex and shows few sequence homologies to other members of the enzyme family.…”

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

“…Angemeldet | 129.187.254.47 Heruntergeladen am | 18.11.13 11:14 sphere can redirect the iron-oxygen reaction in RNR variants to result in hydrocarbon oxidation, as in MMO (Logan et al, 1998;Baldwin et al, 2001;Kolberg et al, 2005). A remarkable feature of the coupled radical iron centers Fe III Fe III -F208-O • formed in mutant RNR is their unusual stability (more than several weeks at room temperature).…”

“…Recently, mutants Y122H (Kolberg et al, 2005) Figure 6), and also shows smaller hyperfine anisotropy, which is typical for Fe III and not for Fe IV (Sturgeon et al, 1996). The paramagnetic center in Y122H was shown to be made up by two Fe III high-spin ions (Ss 5 / 2 ) and a strongly coupled, most probably coordinated radical (Ss 1 / 2 ), which couples to a total spin of Ss 1 / 2 .…”

“…• using phenylalanine-d 8 isotope labeling and mass spectrometry (Kolberg et al, 2005). There is a high structural similarity between the diiron centers in R2 of RNR and in the hydroxylase subunit of methane monooxygenase (MMO), for which reaction of the diferrous iron center with molecular oxygen leads to hydroxylation of the substrate methane (Baik et al, 2003).…”

Advanced EPR Methods for Studying Conformational Dynamics of Nucleic Acids

“…In another case, where the dimer structure was already known from X-ray crystallography, a fine-tuning of the tyrosyl molecule in its radical state in the protein environment was possible, and the results were in perfect agreement with ENDOR experiments performed on single crystals (Kolberg et al, 2005).…”