Evidence by Site-Directed Mutagenesis Supports Long-Range Electron Transfer in Mouse Ribonucleotide Reductase

Abstract

Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

“…5,6). Site-directed mutagenesis has been employed to provide evidence that these residues are essential for radical initiation [9][10][11][12][13] . However, these mutants have not been mechanistically informative, as they exhibit only marginal catalytic activity probably associated to residual wild-type contamination of the purified mutants.…”

Restoring Proper Radical Generation by Azide Binding to the Iron Site of the E238A Mutant R2 Protein of Ribonucleotide Reductase fromEscherichia coli

“…The specific substrate reaction takes place in the larger R1 component, whereas the free radical on a tyrosyl side chain required for enzyme activity is generated in the smaller R2 component on Tyr-122 (1-3). It has been proposed that the substrate binding site in R1 is connected to Tyr-122 in the R2 component by an array of conserved hydrogen-bonded amino acids (4 -6), considered to take part in a coupled electron/proton (H ⅐ radical) transfer between the two proteins during the enzymatic reaction (2,(7)(8)(9)(10). The tyrosyl radical is formed together with a -oxo-bridged diferric center in a reconstitution reaction involving ferrous iron and molecular oxygen.…”

Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei

“…1). The iron ligands (Asp-84, Glu-115, His-118, Glu-204, Glu-238, His-241) (6,46), the tyrosyl free radical (Tyr-122) (5,6,46), the residues in the hydrophobic pocket surrounding the radical (Phe-208, Phe-212, Ile-234) (6,46), and the residues that are proposed to participate in the long-range electron transport pathway (Trp-48, Asp-237, Glu-350, Tyr-356) (6,8,9,46), are all conserved (E. coli class Ia numbering is used throughout the text unless stated otherwise). A striking difference between the mouse and the T. brucei R2 proteins is that the mouse R2 protein is larger due to 53 extra residues in the N terminus.…”

“…The large R1 subunit binds substrates and allosteric effectors, while the small R2 subunit contains a tyrosyl radical (5), generated by a binuclear ferric iron center. The tyrosyl radical in the R2 protein is linked to the active site in the R1 subunit through a hydrogen-bonded long-range electron transport chain (6)(7)(8)(9)(10). Class II RNR generates a radical from 5Ј-deoxyadenosylcobalamin (11,12) and class III RNR, which only works under anaerobic conditions, contains a stable glycyl radical (13).…”