Fourier transform infrared spectroscopy and electrochemistry of the primary electron donor in Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers: vibrational modes of the pigments in situ 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 [Mäntele, 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.(ABSTRACT TRUNCATED AT 250 WORDS)