Characterization of a Rhodobacter capsulatus reaction center mutant that enhances the distinction between spectral forms of the initial electron donor

A large scale mutation of the Rhodobacter capsulatus reaction center M-subu nit gene, sym2-1, has been constructed in which amino acid residues M205-M2 10 have been changed to the corresponding L subunit amino acids. Two interc onvertable spectral forms of the initial electron donor are observed in iso lated reaction centers From this mutant. Which conformation dominates depen ds 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 purifica tion in the presence of high salt. However, upon subsequent dialysis agains t a low ionic strength buffer or the addition of positively charged deterge nts, the near-infrared spectral band of P (the initial electron donor) in s ym2-1 reaction centers is shifted by over 30 nm to the blue, from 852 to 82 0 nm. Systematically varying either the ionic strength or the amount of cha rged detergent reveals an isobestic point in the absorbance spectrum at 845 nm. The wild-type spectrum also shifts with ionic strength or detergent wi th an isobestic point at 860 nm. The large spectral separation between the two dominant conformational forms of the sym2-1 reaction center makes detai led measurements of each state possible. Both of the spectral forms of P bl each in the presence of light. Electrochemical measurements of the P/P+ mid point 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 th e spectral characteristics of P are not critical for charge separation. The short-wavelength form of P in this mutant also converts to the long-wavele ngth 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 mut ant. A similar, though considerably less pronounced spectral change with te mperature occurs in wild-type reaction centers, with an isobestic point at about 855 nm, dose to that found by titrating with ionic strength or deterg ent. Fitting the temperature dependence of the sym2-1 reaction center spect rum to a thermodynamic model resulted in a value for the enthalpy of the co nformational interconversion between the short- and long-wavelength forms o f 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 tem perature dependence in wild-type. Thus, much of the temperature dependence of the reaction center special pair near-infrared absorbance band can be de scribed as an equilibrium shift between two spectrally distinct conformatio ns of the reaction center.