Kinetics of transient radicals in Escherichia coli ribonucleotide reductase. Formation of a new tyrosyl radical in mutant protein R2.
Reconstitution of the tyrosyl radical in ribonucleotide reductase protein R2 requires oxidation of a diferrous site by oxygen. The reaction involves one externally supplied electron in addition to the three electrons provided by oxidation of the Tyr-122 side chain and formation of the mu-oxo-bridged diferric site. Reconstitution of R2 protein Y122F, lacking the internal pathway involving Tyr-122, earlier identified two radical intermediates at Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a novel internal transfer pathway (Sahlin, M., Lassmann, G., Pötsch, S., Sjöberg, B. -M., and Gräslund, A. (1995) J. Biol. Chem. 270, 12361-12372). Here, we report the construction of the double mutant W107Y/Y122F and its three-dimensional structure and demonstrate that the tyrosine Tyr-107 can harbor a transient, neutral radical (Tyr-107(.)). The Tyr-107(.) signal exhibits the hyperfine structure of a quintet with coupling constants of 1.3 mT for one beta-methylene proton and 0.75 mT for each of the 3 and 5 hydrogens of the phenyl ring. Rapid freeze quench kinetics of EPR-visible intermediates reveal a preferred radical transfer pathway via Trp-111, Glu-204, and Fe-2, followed by a proton coupled electron transfer through the pi-interaction of the aromatic rings of Trp-(Tyr-)107 and Trp-111. The kinetic pattern observed in W107Y/Y122F is considerably changed as compared with Y122F: the Trp-111(.) EPR signal has vanished, and the Tyr-107(.) has the same formation rate as does Trp-111(.) in Y122F. According to the proposed consecutive reaction, Trp-111(.) becomes very short lived and is no longer detectable because of the faster formation of Tyr-107(.). We conclude that the phenyl rings of Trp-111 and Tyr-107 form a better stacking complex so that the proton-coupled electron transfer is facilitated compared with the single mutant. Comparison with the formation kinetics of the stable tyrosyl radical in wild type R2 suggests that these protein-linked radicals are substitutes for the missing Tyr-122. However, in contrast to Tyr-122(.) these radicals lack a direct connection to the radical transfer pathway utilized during catalysis.
Katterle
B
B
Department of Molecular Biology, Stockholm University, S-10691 Stockholm, Sweden.
Sahlin
M
M
Schmidt
P P
PP
Pötsch
S
S
Logan
D T
DT
Gräslund
A
A
Sjöberg
B M
BM
D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D014443:Tyrosine
D005609:Free Radicals; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D012264:Ribonucleotide Reductases