Tryptophan and Tyrosine Radicals in Ribonucleotide Reductase:  A Comparative High-Field EPR Study at 94 GHz

Abstract

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

“…For the other tensor components of these protons, values of A x ϭ Ϫ0.9 mT and A y ϭ Ϫ0.35 mT were used, as they are known from other tyrosine radicals (Table II). This assumption is justified because the hyperfine values for these ring protons are similar for the majority of protein tyrosine radicals observed so far (34,(37)(38)(39)(40)(41). The largest hyperfine coupling in tyrosine radicals, which is almost isotropic, results from one of the ␤-protons of the tyrosine side chain.…”

“…The deviation ⌬g x,y ϭ g x,y Ϫ g e from the free electron g value 2.002319 results from spin-orbit coupling and increases when significant spin densities occur at heavier nuclei such as oxygen, which exhibits a larger spin-orbit coupling constant as compared with carbon or nitrogen (42). ⌬g x values are ϳ0.0015 for tryptophan radicals (34), having spin density only on carbon and nitrogen nuclei, whereas for radicals with spin density on oxygen nuclei, such as tyrosines and quinones, ⌬g x values range from 0.004 up to 0.007 (34, 43-45) (Table I) .00219, g y ϭ 2.0042-2.0044; the g x distribution was modeled by the sum of four simulated spectra using g x values between 2.0078 and 2.0062; hyperfine tensors from two ring protons and one ␤-proton (A iso (H ␤1 ) ϭ 1.1 mT) of the side chain (see Table II). Single component line width, 0.6 mT.…”

“…They were also found to be transient intermediates in a variety of enzymes, such as peroxidases (31), cytochrome c oxidase (49), and in DNA photolyase, where, depending on the organism, tryptophan or tyrosine radicals have been observed (50). The reported g x values of tyrosine radicals range from 2.0091 for a non-polar environment in RNR of E. coli (34, 38, 51) to 2.0076 for a hydrogenbonded situation in RNR of mouse and yeast (34,38,39) and in plant photosystem II (37,43). Recently, for a tyrosine radical in turnip peroxidase, a significantly lower value of 2.0066 was reported, probably indicating strong hydrogen bonding (31).…”

“…RNR of E. coli, to 2.0076 for a hydrogen-bonded situation, e.g. for Y D * in plant photosystem II (34,38,53,54) (Table I). For the tyrosine radical recently observed in turnip peroxidase, a distribution of g x values has been reported that is centered around 2.0066 and has a width of ⌬g x ϭ 0.0007 (31).…”

“…where BЈ and BЉ are empirical constants (BЈ Ϸ 0, BЉ ϭ 5.0 -5.24 mT) and is the dihedral angle between the axis of the -orbital (p z ) of the adjacent carbon atom and the projected C ␤ H ␤ bond (27,34,56,57) (Fig. 5B).…”