Selective 2H-, 13C-, and 17O-isotope labeling of the tyrosine amino acid has been used to map the unpaired π-electron spin-density distribution of the UV-generated neutral l-tyrosine phenoxy radical in alkaline frozen solution. The use of 13C and 17O labels allowed accurate determination of the full spin-density distribution and provided more insight in the geometrical structure of the neutral tyrosine radical in vitro. Simulations of the X-band (9.2 GHz) and Q-band (34.8 GHz) EPR powder spectra yielded the principal components of the 1H-, 13C-, and 17O-hyperfine tensors. For the two β-methylene hydrogens, a static conformational distribution of the dihedral angles (90° < θ1 < 60° and 60° < θ2 < 30°) was taken into account. The major proton hyperfine interactions and the principal g values for the neutral tyrosine radical, obtained from selectively deuterated samples, are consistent with literature values. The spin density at the specifically labeled postitions (C1‘, C2‘, C3‘, C4‘, C5‘, O4‘) was evaluated from the anisotropy of the 13C- and 17O-hyperfine tensors. A quantitative analysis of the positions C3‘ and C5‘ provided evidence for a planar distortion of the aromatic ring at these positions. 17O enrichment of the phenol oxygen O4‘ of the tyrosine radical unambiguously showed that the spin density at this oxygen is 0.26 ± 0.01. From the relatively large delocalization of the spin density over the carbonyl group of the tyrosine aromatic ring system, it is concluded that the C4‘−O4‘ bond has a double-bond character. The experimentally determined spin-density distribution is compared with several computational calculated spin-density distributions found in the literature. The isotropic 13C-hyperfine interactions are discussed in the framework of the Karplus−Fraenkel theory. This theory proved to be accurate for the determination of sign and magnitude of the isotropic 13C- and 17O-hyperfine interactions.

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It was previously shown in the photosystem II membrane preparation DT-20 that photoxidation of the oxygen-evolving manganese cluster was blocked by 0.1 mM formate, unless 0.2 mM bicarbonate was present as well [Wincencjusz, H., Allakhverdiev, S. I., Klimov, V. V., and Van Gorkom, H. J. (1996) Biochim. Biophys. Acta 1273, 1-3]. Here it is shown by measurements of EPR signal II that oxidation of the secondary electron donor, YZ, is not inhibited. However, the reduction of is greatly slowed and occurs largely by back reaction with reduced acceptors. Bicarbonate is shown to prevent the loss of fast electron donation to . The release of about one or two free Mn2+ per photosystem II during formate treatment, and the fact that these effects are mimicked by Mn-depletion, suggests that formate may act by replacing a bicarbonate which is essential for Mn binding. Irreversible light-induced rebinding in an EPR-silent form of Mn2+ that was added to Mn-depleted DT-20 was indeed found to depend on the presence of bicarbonate, as did the reconstitution in such material of both the fast electron donation to and the UV absorbance changes characteristic of a functional oxygen-evolving complex. It is concluded that bicarbonate may be an essential ligand of the functional Mn cluster.