FOURIER-TRANSFORM INFRARED DIFFERENCE SPECTROSCOPY OF SECONDARY QUINONE ACCEPTOR PHOTOREDUCTION IN PROTON-TRANSFER MUTANTS OF RHODOBACTER-SPHAEROIDES

In order to investigate the changes of protonation or environment of carboxylic residues occurring upon photoreduction of the secondary quinone acceptor (Q(B)) in the reaction center (RC) of the photosynthetic bacteria Rhodobacter sphaeroides 2.4.1., we have performed light-induced Fourier transform infrared (FTIR) spectroscopy on RCs from wild-type (Wt) and several site-directed mutants. The FTIR Q(B)(-)/Q(B) spectra have been obtained at pH 7 upon single-saturating flash excitation for native RCs and RC mutants containing either a single-site mutation, with Gin at L212 (EQ L212), Asn at L213 (DN L213), or Asn at L210 (DN L210), or a double-site mutation with both Gln at L212 and Asn at L213 (EQ L212 + DN L213). The assignment of an IR band to the protonation/deprotonation of a particular carboxylic side chain was analyzed by combining the effects of site-directed mutagenesis and H-1/H-2 isotope exchange. A positive band at 1728 cm(-1) in the Q(B)(-)/Q(B) spectra was observed in Wt, DN L213, and DN L210 and was absent in the mutants EQ L212 and EQ L212 + DN L213. The intensity of the 1728 cm(-1) band was significantly reduced in (H2O)-H-2, and a new feature appears at 1717 +/- 1 cm(-1). Furthermore, the amplitude of the 1728 cm(-1) band was similar in native and DN L210 RCs but was increased in DN L213. This band is attributed to partial proton uptake by Glu L212 estimated to be 0.3-0.4 H+/Q(B)(-) in native and DN L210 RCs and 0.5-0.6 H+/Q(B)(-) in DN L213 RCs. In contrast, the FTIR Q(B)(-)/Q(B) spectra show no evidence for change of protonation or environment of Asp L213 upon Q(B)(-) formation. The increased protonation of Glu L212 in DN L213 RCs is explained by a decreased Glu L212 pK(a) value due to the loss of a negatively charged Asp L213. Part of a small differential signal at 1732 (+)/1740 (-) cm(-1) that is affected by H-1/H-2 exchange is tentatively assigned to an environmental shift of the protonated Asp L210. A negative signal at 1685 cm(-1) is proposed to arise from the absorption change of the amide I carbonyl mode of Glu L212. The most important conclusions from these FTIR studies are that, after the first electron transfer step which forms Q(B)(-), (1) Glu L212 increases its protonation state, consistent with its proposed function as a proton donor to the fully reduced quinone, and (2) Asp L213 is proposed to be in the ionized state, consistent with the increased proton uptake of Glu L212 in DN L213 RCs and with its function to facilitate proton transfer by creating a negative electrostatic potential near Q(B).