MODIFICATION OF A HYDROGEN-BOND TO A BACTERIOCHLOROPHYLL-A MOLECULE IN THE LIGHT-HARVESTING 1-ANTENNA OF RHODOBACTER-SPHAEROIDES

Site-directed mutagenesis has been used to examine the function of a highly conserved aromatic residue, alpha Trp43, in the light-harvesting 1 antenna of the photosynthetic bacterium Rhodobacter sphaeroides. In this antenna alpha Trp43 is thought to be located near the putative binding site for bacteriochlorophyll; in this work it was changed to both Tyr and Phe, and in each case the main near-infrared absorbance peak was shifted to the blue, from 876 nm to 865 nm and then to 853 nm, respectively. Resonance Raman spectroscopy of the resulting complexes shows a shift of one component of the 1640-cm(-1) peak to 1632 cm(-1) for the Tyr mutant and to 1660 cm(-1) for the Phe mutant. This demonstrates a strengthening of an existing H bond for the Tyr change and a breakage of this bond for the change to Phe. The 1640-cm(-1) peak has been previously assigned to H-bonded C2 acetyl carbonyl groups of both bacteriochlorophylls in the light-harvesting 1 antenna dimer [Robert, B, and Lutz, M. (1985) Biochim. Biophys. Acta 807, 10-21]. These results indicate that one of these H bonds is to alpha Trp43, placing this residue in close proximity to the bacteriochlorophyll alpha macrocycle with which it interacts. The existence of this bond places constraints on the conformation of the cu polypeptide, and a model of an alpha beta heterodimer is presented incorporating these data.