Characterization of a Rhodobacter capsulatus Reaction Center Mutant that Enhances the Distinction between Spectral Forms of the Initial Electron Donor

Abstract

A large scale mutation of the Rhodobacter capsulatus reaction center M-subunit gene, sym2-1, has been constructed in which amino acid residues M205-M210 have been changed to the corresponding L subunit amino acids. Two interconvertable spectral forms of the initial electron donor are observed in isolated reaction centers from this mutant. Which conformation dominates depends on ionic strength, the nature of the detergent used, and the temperature. Reaction centers from this mutant have a ground-state absorbance spectrum that is very similar to wild-type when measured immediately after purification in the presence of high salt. However, upon subsequent dialysis against a low ionic strength buffer or the addition of positively charged detergents, the near-infrared spectral band of P (the initial electron donor) in sym2-1 reaction centers is shifted by over 30 nm to the blue, from 852 to 820 nm. Systematically varying either the ionic strength or the amount of charged detergent reveals an isobestic point in the absorbance spectrum at 845 nm. The wild-type spectrum also shifts with ionic strength or detergent with an isobestic point at 860 nm. The large spectral separation between the two dominant conformational forms of the sym2-1 reaction center makes detailed measurements of each state possible. Both of the spectral forms of P bleach in the presence of light. Electrochemical measurements of the P/P+ midpoint potential of sym2-1 reaction centers show an increase of about 30 mV upon conversion from the long-wavelength form to the short-wavelength form of the mutant. The rate constant of initial electron transfer in both forms of the mutant reaction centers is essentially the same, suggesting that the spectral characteristics of P are not critical for charge separation. The short-wavelength form of P in this mutant also converts to the long-wavelength form as a function of temperature between room temperature and 130 K, again giving rise to an isobestic point, in this case at 838 nm for the mutant. A similar, though considerably less pronounced spectral change with temperature occurs in wild-type reaction centers, with an isobestic point at about 855 nm, close to that found by titrating with ionic strength or detergent. Fitting the temperature dependence of the sym2-1 reaction center spectrum to a thermodynamic model resulted in a value for the enthalpy of the conformational interconversion between the short- and long-wavelength forms of about -6 kJ/mol and an entropy of interconversion of about -35 J/(K mol). Similar values of enthapy and entropy changes can be used to model the temperature dependence in wild-type. Thus, much of the temperature dependence of the reaction center special pair near-infrared absorbance band can be described as an equilibrium shift between two spectrally distinct conformations of the reaction center.

Switching sides—Reengineered primary charge separation in the bacterial photosynthetic reaction center

“…3C). Changes in RC spectral and kinetic properties with conditions (e.g., detergent and temperature) are well known (26)(27)(28)(29). However, to our knowledge, dependence of RC photochemistry on culture density has not been reported.…”

Dynamics of the Excited State in Photosynthetic Bacterial Reaction Centers

“…The finding that the HOMO concentrates largely on P L but the LUMO on P M ,,,, reflects this mixing. The reaction center’s long-wavelength band peaks about 1000 cm –1 to the red of the Q y band of monomeric bacteriochlorophyll at room temperature and has the unusual property of moving further to the red at low temperatures. − Although exciton interactions undoubtedly play a major role in determining the position of the band, and the temperature dependence of the spectrum could possibly be explained by effects of thermal contraction or anharmonic vibrational modes on these interactions, , mixing of exciton and CT transitions probably provides the most economical explanation of these features along with the width and shape of the band and observations from Stark, IR, and hole-burning and linear-dichroism spectroscopy. ,,,− However, this rationalization of the reaction center’s spectroscopic properties requires the 0–0 level of the diabatic P L + P M – CT state to be above the lowest exciton transition in energy.…”

Effects of Ionizable Residues on the Absorption Spectrum and Initial Electron-Transfer Kinetics in the Photosynthetic Reaction Center ofRhodobacter sphaeroides

“…Eastman et al (39) have described small shifts of the P870 band with ionic strength in R. capsulatus RCs. A mutant with replacements of six residues near P, B L , and H L (sym2-1) had a greatly enhanced sensitivity to ionic strength as well as to detergents and temperature.…”