Transient free radicals in iron/oxygen reconstitution of mutant protein R2 Y122F. Possible participants in electron transfer chains in ribonucleotide reductase.
Ferrous iron/oxygen reconstitution of the mutant R2 apoprotein Y122F leads to formation of a diferric center similar to that of the wild-type R2 protein of Escherichia coli ribonucleotide reductase. This reconstitution reaction requires two extra electrons, supplied or transferred by the protein matrix of R2. We observed several transient free radical species using stopped flow and freeze quench EPR and stopped flow UV-visible spectroscopy. Three of the radicals occur in the time window 0.1-2 s, i.e. concomitant with formation of the diferric site. They include a strongly iron-coupled radical (singlet EPR signal) observed only at < or = 77 K, a singlet EPR signal observed only at room temperature, and a radical at Tyr-356 (light absorption at 410 nm), an invariant residue proposed to be part of an electron transfer chain in catalysis. Three additional transient radicals species are observed in the time window 6 s to 20 min. Two of these are conclusively identified, by specific deuteration, as tryptophan radicals. Comparing side chain geometry and distance to the iron center with EPR characteristics of the radicals, we propose certain Trp residues in R2 as likely to harbor these transient radicals.
Sahlin
M
M
Department of Molecular Biology, Stockholm University, Arrhenius Laboratories, Sweden.
Lassmann
G
G
Pötsch
S
S
Sjöberg
B M
BM
Gräslund
A
A
D001059:Apoproteins / Q000378:metabolism*; D003080:Cold Temperature; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D017022:Flow Injection Analysis; D005609:Free Radicals / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010100:Oxygen / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays
D001059:Apoproteins; D005609:Free Radicals; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases; D010100:Oxygen