Vibrational spectrum associated with the reduction of tyrosyl radical D* in photosystem II: a comparative biochemical and kinetic study.

Photosystem II (PSII) contains a redox-active tyrosine, D. Difference FT-IR spectroscopy can be used to obtain structural information about this species, which is a neutral radical, D*, in the photooxidized form. Previously, we have used isotopic labeling, site-directed mutagenesis, and kinetics to assign a vibrational line at 1477 cm-1 to D*; these studies were performed on highly resolved PSII preparations at pH 7.5 ¿Kim et al. (1998) Biochim. Biophys. Acta 1364, 337-360; publisher's correction, Biochim. Biophys. Acta 1366, 330-354¿. Here, we use kinetics to assign vibrational features to tyrosyl radical, D*, in PSII membranes. EPR and fluorescence controls identify a time regime in which D* decay occurs independently of redox changes involving the PSII quinone acceptors. Difference FT-IR spectra, acquired over this time regime, exhibit decreases in the amplitude of a 1477 cm-1 line; quantitative comparison with EPR transients supports the assignment to D*. Conditions, requiring the use of phosphate/formate, have been described for observation of a dissimilar FT-IR spectrum, which has been assigned to tyrosyl radical D*; this spectrum lacks a 1477 cm-1 line ¿Hienerwadel et al. (1997) Biochemistry 36, 14712-14723¿. Under these conditions, we have observed (1) an acceleration in the rate of D* decay and a decrease in D* yield attributable to the presence of formate, (2) a proportional decrease in the amplitude of FT-IR spectra acquired over the time regime in which D* decays, (3) frequency shifts in the D* - D FT-IR spectrum, (4) large-scale structural changes, as assessed by the amide I line shape, and (5) contributions to the FT-IR spectrum from the phosphate/formate buffer in the absence of PSII. We conclude that changes in the FT-IR spectrum, observed in the presence of phosphate/formate, are caused by alterations in the environment of D* and by direct phosphate/formate contributions to the spectrum.