PROBING THE PRIMARY DONOR ENVIRONMENT IN THE HISTIDINEM200-] LEUCINE AND HISTIDINEL173-] LEUCINE HETERODIMER MUTANTS OF RHODOBACTER-CAPSULATUS BY LIGHT-INDUCED FOURIER-TRANSFORM INFRARED DIFFERENCE SPECTROSCOPY

Light-induced P+Q(B)-/PQ(B) FTIR difference spectra of reaction centers (RCs) have been obtained from chromatophores lacking light-harvesting B800-850 antenna for Rhodobacter capsulatus wild type (WT) and for the two mutants His(M200) --> Leu and His(L173) --> Leu. The primary donor (P) in both mutants consists of a bacteriochlorophyll-bacteriopheophytin heterodimer. The most prominent difference between the WT and the mutant spectra is in the 1600-1200-cm-1 region. The WT spectrum displays large positive bands at almost-equal-to 1290, 1500-1430, and 1580-1530 cm-1. These three bands are either small or altogether absent in the heterodimer spectra. In addition, both heterodimer spectra compare well with the electrochemically generated BChla+/BChla spectrum [Mantele, W. G., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472]. These observations indicate that the positive charge is localized on the monomeric BChl in the heterodimers. The overall shape of the ester and keto C=O signals in the BChla+/BChla spectrum is maintained in the in situ spectra although significant differences are observed in the frequency, width, and splitting of the bands. The shape of the signal at 1757/1744 cm-1 in His(L173) --> Leu is comparable to the 1751/1737-cm-1 signal of BChla+/BChla in tetrahydrofuran, indicating a free 10a ester C=O of P(M) in His(L173) --> Leu. The reduced amplitude of the negative 1740-cm-1 feature in both His(M200) --> Leu and WT spectra suggests a hydrogen-bonded 10a ester C=O for P(L). Furthermore, from the striking similarity between the 1751/1740-cm-1 signal of His(M200) --> Leu and WT, a predominant localization of the positive charge on the P(L) side in WT is favored. The frequency range (1683-1696 cm-1) observed for the 9-keto carbonyls of P demonstrates that these groups are free from interaction in situ. An increase in the 9-keto C=O frequency over WT is observed for both P and P+ states in the heterodimers. This upshift and the overall shapes of the differential band are nonequivalent in the two heterodimers, demonstrating that the geometry of the BChl moiety is significantly different in each heterodimer. Thus, the WT spectrum cannot be reconstructed from the two heterodimers' spectra. The differences observed between the heterodimers' spectra and the WT spectrum indicate that the conformation and/or protein local environment of P(L) and P(M) in WT is different from that of P(L) in His(M200) --> Leu and P(M) in His(L173) --> Leu.