Influence of the Axial Ligands on the Spectral Properties of P700 of Photosystem I: A Study of Site-Directed Mutants
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- 作者：Ludwig Krabben ; Eberhard Schlodder ; Rafael Jordan ; Donatella Carbonera ; Giovanni Giacometti ; Hyeonmoo Lee ; Andrew N. Webber ; and Wolfgang Lubitz
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：October 24, 2000
- 年：2000
- 卷：39
- 期：42
- 页码：13012 - 13025
- 全文大小：158K
- 年卷期：v.39,no.42(October 24, 2000)
- ISSN：1520-4995
文摘
Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules whichform the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunitPsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algaeChlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basicamino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulationof PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of theprimary donor characterized. The midpoint potential of P700+
/P700 was increased in all mutants (up to140 mV) and showed a dependence on size and polarizability of the residues when His(B656) wassubstituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additionalbleaching band at 665 nm at room temperature comparable with the published spectrum for the replacementof His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M.,Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance differencespectra of P700+/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectralfeatures around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitutionin PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found inthe 3P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splittingparameters of 3P700 were not significantly changed in the mutated PSI complexes. The electron spindensity distribution of P700+, determined by ENDOR spectroscopy, was only changed when His(B656)was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676).(ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpointpotential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopicfeatures of P700. The data provide convincing evidence that the unpaired electron in the cation radicaland the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinatedby His(B656).
/P700 was increased in all mutants (up to140 mV) and showed a dependence on size and polarizability of the residues when His(B656) wassubstituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additionalbleaching band at 665 nm at room temperature comparable with the published spectrum for the replacementof His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M.,Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance differencespectra of P700+/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectralfeatures around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitutionin PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found inthe 3P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splittingparameters of 3P700 were not significantly changed in the mutated PSI complexes. The electron spindensity distribution of P700+, determined by ENDOR spectroscopy, was only changed when His(B656)was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676).(ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpointpotential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopicfeatures of P700. The data provide convincing evidence that the unpaired electron in the cation radicaland the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinatedby His(B656).