Two different tryptophan radicals (Wa
•
and Wb
•) with lifetimes of several minutes at
room temperature
are formed during the reconstitution of the diiron center in the
Escherichia coli ribonucleotide reductase
mutant
protein R2 Y122F. Detailed hyperfine parameters are for the first
time determined for protein-linked oxidized neutral
tryptophan radicals. Wa
• is
freeze-trapped and investigated by EPR and ENDOR in protonated and
selectively
deuterated proteins at 20 K. Two hyperfine couplings from the
β-methylene protons, hyperfine tensors of two
α-protons, and the complete nitrogen hyperfine tensor are determined.
Based on the absence of a large hyperfine
coupling from the N−H proton, which would be expected for a cation
radical, and on comparison of the experimental
data with theoretical spin densities from density functional
calculations, Wa
• is assigned to an oxidized
neutral
tryptophan radical. A small anisotropic hyperfine coupling
detected in selectively deuterated Wa
• is
tentatively assigned
to a proton which is hydrogen bonded to the nitrogen of
Wa
•. A similar spin density distribution
as for Wa
• is
obtained also for the second tryptophan radical,
Wb
•, observed by EPR at room temperature,
which is also assigned
to an oxidized neutral radical.
A comparative study of X-band EPR and ENDOR of the S2 state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S2 state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S2 state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. (1)H-ENDOR experiments performed at two different field positions of the S2 state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382-2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D2O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S2.
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