Assignment of the Qy absorbance bands of photosystem II chromophores by low-temperature optical spectroscopy of wild-type and mutant reaction centers.
Photosystem II (PSII) contains a collection of pheophytins (Pheo) and chlorophylls (Chl) that have unique absorbance spectra depending on their electronic structure and the surrounding protein environment. Despite numerous efforts to identify the spectra of each cofactor, differing assignments of the chromophore absorbance bands and electrochromic effects have led to conflicting models of pigment organization and chromophore interactions in PSII. We have utilized low-temperature measurements on well-defined redox states, together with the use of site-directed mutants, to make spectral assignments of several reaction center (RC) chromophores. Cryogenic (77 K) optical spectroscopy has been used to trap the bound redox-active quinone, Q(A), in the reduced form and measure the effect of the redox state of Q(A) on PSII chromophores without interference from other redox-active cofactors. The Q(A)(-) minus Q(A) difference spectrum contains a number of features that represent the perturbation of Pheo and Chl absorbance bands upon Q(A) reduction. Using site-directed mutants in which the axial ligand of the D1-side monomeric core Chl, P(A), is changed (D1-H198Q) or the hydrogen-bonding environment of the D1-side Pheo is modified (D1-Q130E), we have assigned the Q(y)() absorbance bands of four chromophores shifted by Q(A) reduction including both RC Pheos, the D1-side monomeric accessory Chl (B(A)), and one other Chl in PSII. The absorbance maximum of B(A) was identified at 683.5 nm from least-squares fits of the D1-H198Q minus wild type (WT) Q(A)(-) minus Q(A) double-difference spectrum; this assignment provides new evidence of a secondary effect of site-directed mutation on a RC chromophore. The other chromophores were assigned from simultaneous fits of the WT and D1-Q130E spectra in which the parameters of only the D1-side Pheo were allowed to vary. The D1-side and D2-side Pheos were found to have lambda(max) values at 685.6 and 669.3 nm, respectively, and another Chl influenced by Q(A)(-) was identified at 678.8 nm. These assignments are in good agreement with previous spectral analyses of intact PSII preparations and reveal that the number of chromophores affected by Q(A) reduction has been underestimated previously. In addition, the assignments are generally consistent with chromophore positions that are similar in the PSII RC and the bacterial photosynthetic RC.
Stewart
D H
DH
Sterling Chemistry Laboratory, Yale University, P.O. Box 208107, New Haven, Connecticut 06520-8107, USA.
Nixon
P J
PJ
Diner
B A
BA
Brudvig
G W
GW
D001429:Bacteriochlorophylls / Q000737:chemistry / Q000235:genetics; D016227:Benzoquinones / Q000737:chemistry*; D003080:Cold Temperature; D000458:Cyanobacteria / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D005615:Freezing; D005973:Glutamine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D045342:Light-Harvesting Protein Complexes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics*; D045332:Photosystem II Protein Complex; D013053:Spectrophotometry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics
D001429:Bacteriochlorophylls; D016227:Benzoquinones; D005609:Free Radicals; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C066447:tyrosyl radical Y(D); C114979:tyrosyl radical YZ; D005973:Glutamine; C004532:quinone; D014443:Tyrosine; D006639:Histidine