Photosystem II, the photosynthetic water oxidizing complex, contains two well characterized redox active tyrosines, D and Z. D forms a stable radical of unknown function. Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site. The vibrational difference spectra associated with the oxidation of tyrosines Z and D have been obtained through the use of infrared spectroscopy (MacDonald, G. M., Bixby, K.A., and Barry, B.A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11024-11028). Here, we examine the effect of deuterium exchange on these vibrational difference spectra. While the putative C-O vibration of stable tyrosine radical D. downshifts in 2H2O, the putative C-O vibration of tyrosine radical Z. does not. This result is consistent with the existence of a hydrogen bond to the phenol oxygen of the D. radical; we conclude that a hydrogen bond is not formed to the Z. radical. In an effort to identify the amino acid residue that is the proton acceptor for Z, we have performed global 15N labeling. While significant 15N shifts are observed in the vibrational difference spectrum, substitution of a glutamine for a histidine that is predicted to lie in the environment of tyrosine Z has little or no effect on the difference infrared spectrum. There is also no significant change in the yield or lineshape of the Z. EPR signal under continuous illumination in this mutant. Our results are inconsistent with the possibility that this residue, histidine 190 of the D1 polypeptide, acts as the sole proton acceptor for tyrosine Z.

Photosystem II, the photosynthetic wateroxidizing complex, contains two redox-active tyrosine residues.Although current models suggest that these tyrosines are located in symmetric positions in the reaction center, there are functional differences between them. To elucidate those structural factors that give rise to this functional asymmetry, we have used difference Fourier-transform infrared spectroscopy to obtain the vibrational difference spectrum associated with the oxidation of each of these redox-active residues. Isotopic labeling was employed to definitively assign vibrational lines to the redox-active tyrosines. This work has shown that the vibrational spectra of the two redox-active species are significantly different from each other. This result suggests that the structure of the redox-active residue helps to determine its role in electron transfer in the reaction center.Photosystem II (PSII) is the photosynthetic membrane complex that carries out the light-induced oxidation of water and reduction of plastoquinone in plants, green algae, and prokaryotic cyanobacteria. Isotopic labeling and electron paramagnetic resonance (EPR) spectroscopy have been used to show that PSII contains two redox-active tyrosine residues (1, 2). One of these tyrosines, Z, is involved in electron transfer between the primary chlorophyll donor, P680, and the manganese-containing catalytic site (for review, see ref.3). The other tyrosine, D, is not directly involved in the electron-transfer events that lead to oxygen evolution; the function of D is not known (4). These tyrosines are believed to be symmetrically located in the reaction center (5-8).The oxidized forms of D and Z, D-and Z-, are neutral radicals that give rise to characteristic and similar EPR signals (1, 2). These EPR signals have been used to show that D and Z have different oxidation and reduction kinetics. Z is oxidized by P680+ (9), and Z-is, in turn, reduced by the manganese cluster (10, 11). If the metal cluster is removed, the reduction of Z-is slowed to the millisecond time regime (12)(13)(14). On the other hand, D is usually oxidized by the manganese cluster with a 1-sec rise time (15). In the absence of a functional manganese cluster, D can be oxidized by P680+ (16, 17). The decay time of D-is on the order of several hours (18).In addition to the kinetic differences described above, there are other functional differences between the Z and D tyrosines. For example, the redox potential of D has been measured to be 0.76 V (19), whereas the potential of Z has been estimated to be about 1 V (20). Further, the two radicals show different accessibility to exogenous reductants (21).This functional asymmetry may be due to differences in the structure of the two redox-active residues. The EPR spectra of Z-and D-do not give detailed insight into this question. Since these radicals are immobilized, their EPR lineshapes are broadened by anisotropic interactions, and small structural alterations would not be readily observable. Recently,The publication costs o...

There are two redox-active tyrosines in photosystem II, the water-splitting complex, that form neutral tyrosine radicals. One of these tyrosine radicals, D., is stable and has an unknown function. The other redox-active tyrosine, Z, acts to transfer oxidizing equivalents from the primary chlorophyll donor of photosystem II to the manganese cluster, where water oxidation occurs. In an attempt to obtain more information about Z and its interaction with its environment, we have begun a study using Fourier-transform infrared (FT-IR) vibrational spectroscopy. To facilitate these studies, we have developed a procedure to isolate spinach photosystem II complexes with an antenna size of approximately 100-110 chlorophylls per reaction center. These complexes show an approximately 2-fold increase in the specific activity of oxygen evolution over the activity of the starting material, photosystem II membranes. Although fully solubilized in detergent, these complexes retain the 24- and 18-kDa extrinsic proteins and exhibit no calcium chloride requirement for optimal oxygen evolution. In manganese-depleted photosystem II samples, Z. can be accumulated in the light. In the dark, the tyrosine radical is reduced and reprotonated to form the neutral tyrosine. Since this process is reversible and light-dependent, we have used light-minus-dark difference FT-IR spectroscopy to observe the vibrational difference spectrum that is associated with the oxidation of this residue. As a control, EPR spectra were measured under identical conditions to assess the amount of Z. that accumulated in the light. We also hope to use difference FT-IR to identify the amino acid with which Z may form a hydrogen bond.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a(3)-Cu(B) binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu(B). Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu(B) and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3). Furthermore, using time-resolved (5 micros resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa(3) and bo(3). In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu(B)-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O(2) in flow-flash experiments.