Changes in primary donor hydrogen-bonding interactions in mutant reaction centers from Rhodobacter sphaeroides: identification of the vibrational frequencies of all the conjugated carbonyl groups.
Specific changes in the hydrogen-bonding states of the primary donor, P, in reaction centers from Rhodobacter sphaeroides bearing mutations near P were determined using near-infrared excited Fourier transform (FT) Raman spectroscopy. This technique, using 1064-nm excitation, provides the preresonantly enhanced vibrational spectrum of P in its reduced state selectively over the contributions of the other reaction center chromophores and protein and yields structural information concerning P and its hydrogen-bonding interactions. The mutations studied were as follows: Leu M160-->His, Leu L131-->His, the D9 double mutant (Leu M160-->His + Leu L131-->His), Phe M197-->His, and His L168-->Phe. These mutations were designed to introduce new, or to break existing, hydrogen bonds to the C9 and C2 carbonyl groups of P. On the basis of previous assignments [Mattioli, T. A., Hoffmann, A., Robert, B., Schrader, B., & Lutz, M. (1991) Biochemistry 30, 4648-4654], the FT Raman spectra of these mutants show the predicted changes in hydrogen bond interactions of P carbonyl groups with the protein. The results of this study have permitted us to unambiguously identify the C2 and C9 carbonyl vibrators of P in Rb. sphaeroides. The genetically introduced hydrogen bond interactions are discussed in terms of other physicochemical properties of P including the redox potential and electronic asymmetry in the P+ state. It is discussed that changes in protein hydrogen bonding to the conjugated carbonyl groups of P alone are not the sole factor that contributes to the sizeable modifications of the P/P+ redox midpoint potentials, and that the chemical nature of the hydrogen bond donor plays a significant role in this modification.
Mattioli
T A
TA
Département de Biologie Cellulaire et Moléculaire, CEA and URA CNRS 1290, C. E. Saclay, Gif-sur-Yvette, France.
Williams
J C
JC
Allen
J P
JP
Robert
B
B
D002244:Carbon / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006639:Histidine; D006860:Hydrogen Bonding; D007930:Leucine; D015394:Molecular Structure; D016296:Mutagenesis; D009154:Mutation; D010649:Phenylalanine; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship
D045322:Photosynthetic Reaction Center Complex Proteins; D010649:Phenylalanine; D006639:Histidine; D002244:Carbon; D007930:Leucine