RESUMEN
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.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter capsulatus , Secuencia de Aminoácidos , Transporte de Electrón , Concentración de Iones de Hidrógeno , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Concentración Osmolar , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Potenciometría , EspectrofotometríaRESUMEN
Nine large-scale symmetry reaction center mutants were constructed in Rhodobacter capsulatus by replacing segments of the M subunit gene with the homologous region of the L subunit gene. Between them, the mutations resulted in symmetrization of essentially the entire region from the carboxy terminal portion of the C helix through most of the E helix. The amino acids in this region define about 80% of the environment of the reaction center cofactors. These studies show that roughly 80% of the amino acids that come in close contact with the cofactors involved in initial electron transfer can be made symmetric in a piecewise manner without loss of the ability to grow photoheterotrophically. However, the amino acid regions near the quinones and iron atom are much more sensitive to symmetrization and most of the large-scale changes in this region resulted in the loss of photosynthetic viability, probably due to loss of stable reaction centers from the photosynthetic membrane. More detailed analysis of the isolated photosynthetic membranes from these mutants showed that in all cases but one, there was some amount of charge separation occurring in the mutant reaction centers. This bank of mutants serves as a useful starting point for more detailed studies of the differential molecular interactions which occur between the two reaction center subunits and their associated cofactors.
Asunto(s)
Proteínas Bacterianas , Fotosíntesis/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter capsulatus/genética , Secuencia de Aminoácidos , Fluorescencia , Genes Bacterianos , Luz , Datos de Secuencia Molecular , Mutagénesis , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Potenciometría , Espectrofotometría , Relación Estructura-Actividad , Supresión GenéticaRESUMEN
Reaction centers isolated from three large-scale symmetry mutants sym0, sym2-1, and sym5-2 described in the previous article of this issue [Taguchi, A. K. W., Eastman, J. E., Gallo, D. M., Jr., Sheagley, E.. Xiao, W., & Woodbury, N. W. (1996) Biochemistry 35, 3175-3186] have been investigated by low-temperature ground state and ferntosecond-resolution transient absorption spectroscopy. All three of these large-scale symmetry mutants undergo electron transfer at 20 K. The mutants sym0 and sym5-2 have yields and dominant rates of charge separation comparable to wild type. However. the sym2-mutant shows a roughly 35%, quantum yield at this temperature, and the major kinetic component of the initial electron transfer is slower than wild type by nearly a factor of 100. The sym0 mutant showed substantial changes in the monomer bacteriochiorophyll ground state and transient spectra, and both sym0 sym2-1 showed changes in the bacteriopheophyll ground state and transient spectra. In particular, sym2-1 shows a small absorbance decrease in the region of the Qx band of the B side bacteriopheophytin which could be attributed to 10%-20% electron transfer along the B pathway.