DIRECT EVIDENCE OF STRUCTURAL-CHANGES IN REACTION CENTERS OF RB. SPHAEROIDES CONTAINING SUPPRESSOR MUTATIONS FOR ASP L213-]ASN - A FTIR STUDY OF Q(B) PHOTOREDUCTION

In bacterial reaction centers (RCs), changes of protonation state of c arboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon QB photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to t he detrimental Asp L213 --> Asn replacement on Q(B)(-)/Q(B) FTIR spect ra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 --> Asp, Arg M233 --> Cys, or Arg H177 --> His suppressor m utation. The proton uptake by Glu L212 upon Q(B)(-) formation, as refl ected by the positive band at 1728 cm(-1), is increased in the Asn M44 --> Asp and Arg H177 His suppressor RCs with respect to native RCs, a nd remains comparable to that observed in Asp L213 --> Asn mutant RCs. Only the Arg M233 --> Cys suppressor mutation affected the 1728 cm(-1 ) band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of t he mutant spectra, several protein (amide I and amide II) and quinone anion (C - O/C - C) modes are perturbed compared to the spectrum of na tive RCs. These IR data show that all of the compensatory mutations al ter the semiquinone-protein interactions and the backbone providing di rect evidence of structural changes accompanying the restoration of ef ficient proton transfer in RCs containing the Asp L213 --> Asn lesion.