The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and htdine residues in the reaction center from Rhodobacter sphaeroides have been igaed. Sitedirected mutations were deiged to add or remove hydrogen bonds between the 2-acetyl groups of the dimer and istidine residues at the setry-related sites Hls-L168 and Phe-M197, and between the 9-keto groups and Leu-L131 and Leu-M160. The addition of a hydrogen bond was correlated with an increase in the dimer midpoint potential. Measurements on double and triple mutants showed that changes In the midpoint potential due to alterations at the individual sites were additive.Midpoint potentials ranging from 410 to 765 mV, compared with 505 mV for wild type, were achieved by various combinations of mutations. The optical absorption spectra of the reaction centers showed relatively minor changes in the position of the donor absorption band, indicating that the addiion of hydrogen bonds to hide pimril destabilized the oxidized state of the donor and had little effect on the excited state relative to the ground state. Despite the change in energy of the charge-separated states by up to 260 meV, the mutant reaction centers were still capable of electron taner to the primary quinone. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quin , and a fit of these data using the Marcus equation idicated that the reorgn i energy for this reaction is =400 meV higher than the change in free energy in wild type. The mutants were still capable of photosynthetic growth, although at reduced rates relative to the wild type. These results suggest a role for protein-cofactor interactions-n particular, hididonor interactions-In establishing the redox potentials needed for electron transfer in biological systems.Although the oxidation-reduction midpoint potentials of identical cofactors in redox proteins can vary by several hundred millivolts, the specific interactions of the cofactor with the protein that result in the variation in midpoint potential are not well understood. The primary electron donor in reaction centers from the purple photosynthetic bacterium Rhodobacter sphaeroides is a bacteriochlorophyll (Bchl) dimer designated P (reviewed in refs. 1-3). The two Bchls of the dimer, labeled A and B, overlap in ring I, where they are separated by -3.5 A. The midpoint potential of the primary donor is =500 mV in wild-type reaction centers from Rb. sphaeroides (4-6) and is expected to be a critical parameter for electron transfer reactions that involve the donor, as alteration of the potential will result in a change in the driving force for these reactions.Mutagenesis experiments have shown that hydrogen bonds between histidine residues and the conjugated carbonyls of the Bchls in the dimer can alter the midpoint potential by significant amounts (5, 7-10). For each Bchl there are two groups, the 9-keto group of ring V and the 2-acetyl group of ring I, t...

Mutations were made in four residues near the bacteriochlorophyll cofactors of the photosynthetic reaction center from Rhodobacter sphaeroides. These mutations, L131 Leu to His and M160 Leu to His, near the dimer bacteriochlorophylls, and M203 Gly to Asp and L177 Ile to Asp, near the monomer bacteriochlorophylls, were designed to result in the placement of a hydrogen bond donor group near the ring V keto carbonyl of each bacteriochlorophyll. Perturbations of the electronic structures of the bacteriochlorophylls in the mutants are indicated by additional resolved transitions in the bacteriochlorophyll absorption bands in steady-state low-temperature and time-resolved room temperature spectra in three of the resulting mutant reaction centers. The major effect of the two mutations near the dimer was an increase up to 80 mV in the donor oxidation-reduction midpoint potential. Correspondingly, the calculated free energy difference between the excited state of the primary donor and the initial charge separated state decreased by up to 55 mV, the initial forward electron-transfer rate was up to 4 times slower, and the rate of charge recombination between the primary quinone and the donor was approximately 30% faster in these two mutants compared to the wild type. The two mutations near the monomer bacteriochlorophylls had minor changes of 25 mV or less in the donor oxidation-reduction potential, but the mutation close to the monomer bacteriochlorophyll on the active branch resulted in a roughly 3-fold decrease in the rate of the initial electron transfer.

Two mutations, L168 His to Phe and L167 Phe to Leu, were made in residues near the primary electron donor, a bacteriochlorophyll dimer, of the reaction center from Rhodobacter sphaeroides. Blue shifts of 10-15 nm in the 865-nm band of the donor were observed in the optical absorption spectra of both of the mutant reaction centers. The rate of initial electron transfer was determined by measurement of the kinetics of the decay of the excited state of the donor, and the rate of charge recombination was determined by measurement of the recovery of the bleaching of the donor. The initial electron transfer time constant and the charge recombination time constant were determined to be 3.6 ps and 220 ms, respectively, in the L168 His to Phe mutant and 5.0 ps and 85 ms in the L167 Phe to Leu mutant, compared to 3.8 ps and 100 ms measured for the wild type. The oxidation potential of the donor measured by oxidation-reduction titrations was found to decrease by 80 mV in the L168 His to Phe mutant and increase by 25 mV in the L167 Phe to Leu mutant. Time-resolved fluorescence decay measurements indicated that the change in the oxidation potential of the donor in the L168 His to Phe mutant resulted in a change in the energies of the charge-separated states. The results show that an increase in the driving force does not increase the rate of the initial electron transfer reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

The redox properties, absorption, electroabsorption, CD, EPR, and P+QA- recombination kinetics have been measured for the special pairs of two mutants of Rhodobacter capsulatus reaction centers involving amino acid changes in the vicinity of the special pair, P. Both mutants symmetrize amino acid residues so that portions of the M-sequence are replaced with L-sequence: sym1 symmetrizes all residues between M187 and M203, whereas (M)F195H is a single amino acid subset of the sym1 mutation. (M)F195H introduces a His residue in a position where it is likely to form a hydrogen bond to the acetyl group of the M-side bacteriochlorophyll of P. For both mutants compared with wild-type, (i) the redox potential is at least 100 meV greater, (ii) the P+QA- recombination rate is about twice as fast at room temperature, and (iii) the large electroabsorption feature for the QY band of P is shifted relative to the absorption spectrum. The comparison of the properties observed for the sym1 and (M)F195H reaction center mutants and the differences between these mutants and wild-type suggest that residue M195 is an important determinant of the properties of the special pair.

No abstract