Aileen K. W. Taguchi

The role of reaction center excited state evolution during charge separation in a Rb. sphaeroides mutant with an initial electron donor midpoint potential 260 mV above wild type

Abstract Femtosecond transient absorbance spectroscopy was performed on the triple hydrogen bond reaction center mutant [LH(L131) + LH(M160) + FH(M197)] of Rhodobacter sphaeroides which has a P/P+ midpoint potential 260 mV above wild type. The decay of the excited singlet state in this mutant is kinetically complex with a dominant decay component of about 50 ps at 295 K. Charge separation to the state P+QA− occurs with a quantum yield of 0.50 ± 0.1 at 295 K and 0.10–0.15 at 20 K. The yield, rate of formation and spectra of states which are trapped when electron transfer to the quinone is blocked by quinone reduction compared to the rate and yield of formation of P+QA− in unreduced reaction centers suggest that evolution of the excited state is the rate limiting event in charge separation in triple mutant reaction centers. The excited state that results from this evolution has spectral features which are remarkably similar to the initial excited singlet state found using R-26 reaction centers (R-26 reaction centers have essentially wild type photochemistry). The fact that the formation of this altered excited state is greatly slowed in a high P/P+ midpoint potential mutant suggests that the early excited state in wild type or R-26 reaction centers may have considerable P+ character. A consideration of the thermodynamics of the state P+BA− in this and related high potential mutants implies that a simple model in which P+BA− is formed as a discrete electron transfer intermediate is not a viable description in these mutants. Other factors such as reaction center heterogeneity or alternate electron transfer mechanisms must be invoked.

Genetic Manipulation of Purple Photosynthetic Bacteria

Genetic manipulation has become widely used in the study of purple photosynthetic bacteria. Some species have been extensively characterized genetically and thus are readily amenable to new applications of these techniques, while in other species genetic work is being developed and may require some adaptation of existing protocols. Genetic studies in purple bacteria include both classical genetic mapping that yields information about the genomic distribution of the genetic loci under study and molecular genetic techniques that employ recombinant DNA techniques and center on analysis at a nucleotide level. As a result of these studies, a large number of genes have been characterized, most prominently those whose products are involved in photosynthesis and nitrogen fixation, but also including a variety of other processes. Techniques that have been used to identify genes range from complementation ofmutant strains to hybridization probes based on homologous genes or the amino acid sequences of proteins. The functional properties of genes have been studied by the generation of random mutations by chemical or transposon mutagenesis and by site-directed insertions, deletions and alterations of nucleotide sequences. The transcription and translation of genes have been investigated through gene fusions and in vitro and heterologous expression systems. The combination of classical genetics with the advances in molecular biological techniques provides an array of tools for dissecting the underlying basis for metabolic processes in these bacteria.

Photosynthetic reaction center mutagenesis via chimeric rescue of a non-functional Rhodobacter capsulatus puf operon with sequences from Rhodobacter sphaeroides

Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3′ of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3′ end of pufM and sequences past the end of pufM.

Preliminary Characterization of pAT-3, a Symmetry Enhanced Reaction Center Mutant of Rhodobacter capsulatus

A Rhodobacter capsulatus reaction center mutant has been constructed in which residues M187 through M203 have been replaced by the homologous region of the L polypeptide (L160 through L176). This mutant expresses reaction centers, grows photosynthetically and undergoes electron transfer forming P+QA − with high quantum yield. Absorption and Stark measurements indicate a significant alteration in the electronic nature of the special pair, and time-resolved fluorescence measurements suggest changes in the photochemistry of the initial electron transfer reaction. Finally, the redox potential of the special pair has apparently been increased significantly in this mutant to greater than 500 mV.

Reaction Center Heterogeneity Probed by Multipulse Photoselection Experiments With Picosecond Time Resolution

Using double pulse excitation of an electron-transfer-impaired reaction center mutant from Rhodobacter sphaeroides, it has been possible to provide direct evidence for reaction center conformational heterogeneity which affects the charge separation yield. A saturating prepulse was used to photoselect a subpopulation of reaction centers which returned rapidly to the ground state instead of forming the long-lived P+Q A - state. Femtosecond transient absorbance measurements were then performed on this photoselected subpopulation. It was found that the yield of electron transfer in this subpopulation was substantially lower than that in the bulk reaction center population. Though these measurements are preliminary, they represent the first direct demonstration of a reaction center conformational heterogeneity that clearly affects the efficiency of the initial electron transfer reaction.

Formation of Charge Separated State on the Inactive Side of the R-26 Reaction Center Using High Intensity Excitation

One of the most striking features of the bacterial reaction center is its unidirectional electron transfer. Though three dimensional structures of purple bacterial reaction centers reveal a two-fold symmetric configuration of the proteins and cofactors [e.g. 1, 2], photon initiated electron transfer from the special pair P was found preferentially along only one of the two branches, denoted A. The cofactors involved in the electron transfer chain are BA, HA, and QA, and the first detectable charge separated state is P+HA-. The inactive side of the reaction center is referred to B branch. No detectable FB,- state has been observed spectroscopically so far from wild type reaction centers, except under photopumping conditions [for reviews, see: 3, 4].

Chimeric Rescue of Rb. capsulatus Reaction Center Genes with Sequences from Rb. sphaeroides

Site-directed mutagenesis is becoming a very important technique in the study of photosynthetic reaction center structure-function relationships. However, there are certain disadvantages associated with this approach. First, it relies very heavily on the intuition of the investigator to select particular mutations that will give rise to interesting phenotypes; many times, mutagenesis can result in very little change in photosynthetic function, in complete loss of function, or in lack of assembly. Second, it is difficult to use specific mutagenesis to detect the concerted effect of several amino acids.