FOURIER-TRANSFORM INFRARED-SPECTROSCOPY AND ELECTROCHEMISTRY OF THE PRIMARY ELECTRON-DONOR IN RHODOBACTER-SPHAEROIDES AND RHODOPSEUDOMONAS-VIRIDIS REACTION CENTERS - VIBRATIONAL-MODES OF THE PIGMENTS INSITU AND EVIDENCE FOR PROTEIN AND WATER MODES AFFECTED BY P+ FORMATION

Protein electrochemistry in an ultra-thin-layer electrochemical cell suitable for UV/vis and IR spectroscopy has been used to characterize the vibrational modes of the primary electron donors of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers in their neutral and cation radical states (P and P+, respectively). The P-->P+ redox transitions could be well separated from redox reactions of other cofactors according to their redox midpoint potential. The IR difference bands of the primary electron donor bacteriochlorophylls all titrate in unison and exhibit the correct midpoint potential. Comparison of the difference spectra with those of isolated bacteriochlorophylls a and b in organic solvents of different polarity and proton activity [Mantele, W., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472] leads to similar conclusions on the binding and interaction of the pigments within the protein matrix as previously obtained from light-induced P+Q-/PQ difference spectra. Equilibration of the reaction centers in D2O leads to few but distinct shifts of bands and changes of band intensities at 1662, 1634, and 1526 cm-1 (Rhodobacter sphaeroides) and 1694, 1664, 1648, 1630, and 1532 cm-1 (Rhodopseudomonas viridis) as well as to smaller deviations at other wavenumbers. The H-->D-sensitive band at 1662 cm-1 is interpreted in terms of a histidine NH2+ bending mode. A second H/D-sensitive difference band around 1648 cm-1 in the Rhodopseudomonas viridis reaction center may be associated with the peptide C=O of one of the amino acids surrounding P [eventually of the histidine(s) ligating the Mg] which is affected by P+ formation. Small negative signals at 1526 cm-1 (Rhodobacter sphaeroides) and 1532 cm-1 (Rhodopseudomonas viridis) sensitive to H-D exchange are tentatively assigned to a tyrosine residue, presumably Tyr(M210) (Rhodobacter sphaeroides) and Tyr(M208) (Rhodopseudomonas viridis), and could correspond to a change of hydrogen bonding of the phenyl hydroxy group or to a change of its conformation upon primary charge separation. A broad background signal in the 1620-1670-cm-1 range for Rhodobacter sphaeroides reaction centers disappearing in D2O is assigned to a H-O-H bending mode. Together with two broad band features at 1480-1400 and 1250-1150 cm-1, which can be assigned to the H-O-D and D-O-D bending modes, respectively, it is interpreted in terms of water molecule(s) in the primary donor vicinity affected by primary electron donor oxidation.