Abstract
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.
Highlights
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
Protein R2 has been characterized in a radical-free state, metR2, where the diferric site remains but the tyrosyl radical is reduced to a normal tyrosine residue
Concluding Remarks-The present results demonstrate the transient formation of a large number of EPR and/or optically active paramagnetic species on different time scales when the E. coli protein R2 mutant Y122F reacts with Fe 2+ and 02' In analogy with the corresponding reaction in wild-type R2, we consider the reconstitution to take place between a reduced dinuclear iron center and oxygen
Summary
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 ofR2. The R2 activation reaction (formation of diferric center and tyrosyl radical) has been studied in extensive detail (Peterson et al, 1980; Lynch et al, 1989; Sahlin et al, 1990; Fontecave et al, 1990; Ochiai et al, 1990; Bollinger et al, 1991a, 1994a, 1994b).
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