Primary Quinone (QA) Binding Site of Bacterial Photosynthetic Reaction Centers:  Mutations at Residue M265 Probed by FTIR Spectroscopy

In the primary quinone (QA) binding site of Rb. sphaeroides reaction centers (RCs), isoleucine M265 is in extensive van der Waals contact with the ubiquinone headgroup. Substitution of threonine or serine for this residue (mutants M265IT and M265IS), but not valine (mutant M265IV), lowers the redox midpoint potential of QA by about 100 mV (Takahashi et al. (2001) Biochemistry 40, 1020−1028). The unexpectedly large effect of the polar substitutions is not due to reorientation of the methoxy groups as similar redox potential changes are seen for these mutants with either ubiquinone or anthraquinone as QA. Using FTIR spectroscopy to compare QA -/QA IR difference spectra for wild type and the M265 mutant RCs, we found changes in the polar mutants (M265IT and M265IS) in the quinone CO and CC stretching region (1600−1660 cm-1) and in the semiquinone anion band (1440−1490 cm-1), as well as in protein modes. Modeling the mutations into the X-ray structure of the wild-type RC indicates that the hydroxyl group of the mutant polar residues, Thr and Ser, is hydrogen bonded to the peptide CO of ThrM261. It is suggested that the mutational effect is exerted through the extended backbone region that includes AlaM260, the hydrogen bonding partner to the C1 carbonyl of the quinone headgroup. The resulting structural perturbations are likely to include lengthening of the hydrogen bond between the quinone C1O and the peptide NH of AlaM260. Possible origins of the IR spectroscopic and redox potential effects are discussed.