Theodore J. DiMagno

Initial electron transfer in photosynthetic reaction centers of Rhodobacter capsulatus mutants

Abstract The stimulated emission decay time constants were measured for a series of Rhodobacter capsulatus reaction centers with site-specific mutations at the symmetry related locations M208 and L181. We report the first mutant (Phe L181 → Tyr) that exhibits an initial electron transfer rate faster than the native organism at 295 K, and determine that the tyrosine at position M208 cannot be fully responsible for the unidirectionality of electron transfer.

Protein-chromophore interactions: spectral shifts report the consequences of mutations in the bacterial photosynthetic reaction center

Features of the low temperature absorption spectra of reaction centers from a large family of Rhodobacter capsulatus strains carrying site-specific mutations at the M208Tyr and L181Phe positions are presented. Through systematic analysis of the observed electronic transitions (with accompanying vibronic bands), the primary effects of many of the mutations have been identified and explained in terms of the reaction center structure. Some of the observed shifts are comparable to those that have been seen in other systems as a result of formation of hydrogen bonds or hexacoordination of the central Mg atom in bacteriochlorophyll species. Shifts of the Qy and Qx bacteriochlorophyll bands are also seen as a result of distant mutations near the quinones that change the charge distribution of the reaction center. We include kinetic data which show that the spectral and kinetic characteristics of different cofactors can be modulated depending on the particular amino acid that is substituted at the M208 site.

STUDY OF REACTION CENTER FUNCTION BY ANALYSIS OF THE EFFECTS OF SITE-SPECIFIC AND COMPENSATORY MUTATIONS

The protein portion of the reaction center (RC) complex is composed of three subunits: the intermembrane L and M chains, and the cytoplasmic H polypeptide. The cofactors are within the transmembrane region; they are related by approximate twofold symmetry as are the homologous L and M chains [1–4]. The cofactors consist of a bacteriochlorophyll dimer that is the primary electron donor, two bacteriochlorophyll monomers, two bacteriopheophytins, a non-heme iron atom, and two quinones which serve as the final electron acceptors.

Recent Experimental Results for the Initial Step of Bacterial Photosynthesis

This paper discusses experiments pertaining to two major issues of the primary events of photosynthesis: Why is only a single side (A-side) of the highly C2 symmetrical reaction center active? How does the electron get from the special pair donor (P) to the bacteriopheophytin acceptor (HA)? The investigation presented here highlights the correlation between the reduction potential of the primary donor and the initial charge separation rate measured by femtosecond spectroscopy in a series of mutated reaction centers from Rb. capsulatus 1.

Initial Electron Transfer Events in Photosynthetic Bacteria

This chapter explores the initial electron transfer events in photosynthetic bacteria. Structural determination has provided detailed information about the spatial arrangement of all the cofactors involved in the electron transfer reactions and about their interactions with the protein solvent. The reaction center protein complex is the site at which the ultrafast and unidirectional electron transfer takes place. The initial charge-separation process was believed to occur via a single exponential reaction characterized by a ∼3-ps time constant at room temperature. The initial chemistry is at least bi-exponential, with an 80% amplitude component with a fast time constant of 2.8 ps and a 20% amplitude component with a slower constant of about 11 ps. In the primary event, the electron is transferred from singlet excited state of the special pair donor to the bacteriopheophytin associated with the L subunit. The electron is then transferred to Q A in approximately 200 ps and then to Q B in roughly 100–200 μs. There are three unresolved issues surrounding the primary events of purple bacterial photosynthesis: (1) the unidirectionality of the electron transfer, (2) the role of the accessory bacteriochlorophyll in the initial electron transfer mechanism, and (3) the temperature dependence of the electron transfer reactions. The fast component of the electron transfer rate speeds up as the temperature is lowered, indicating that little or no thermal activation energy is required for this process.

A Structural Basis for Electron Transfer in Bacterial Photosynthesis

Triplet data for the primary donor in single crystals of bacterial reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis are interpreted in terms of the corresponding x-ray structures. The analysis of electron paramagnetic resonance data from single crystals (triplet zero field splitting and cation and triplet linewidth of the primary special pair donor of bacterial reaction centers) is extended to systems of a non-crystalline nature. A unified interpretation based on frontier molecular orbitals concludes that the special pair behaves like a supermolecule in all wild-type bacteria investigated here. However, in heterodimers of Rhodobacter capsulatus (HisM200 changed to Leu or Phe with the result that the M-half of the special pair is converted to bacteriopheophytin) the special pair possesses the EPR properties more appropriately described in terms of a monomer. In all cases the triplet state and cation EPR properties appear to be dominated by the highest occupied molecular orbitals. These conclusions derived from EPR experiments are supplemented by data from Stark spectroscopy of reaction centers from Rb. capsulatus. The most red-shifted Stark band in the Rb. capsulatus heterodimer is relatively intense and is interpreted as a “pure” charge transfer band within the special pair donor. This explanation locates the energy of Bchl- M>Bchl+L> or Bchl+ M>Bchl- L>, the internal charge transfer state, anywhere from ~.1 ev to ~.3 ev above 1*[BchlM>BchlL>], the first excited singlet state of the primary donor. This internal charge transfer state has been invoked previously in the initial mechanism of electron transfer in photosynthesis.

A Magnetic View of the Reaction Center

The understanding of natural photosynthesis has advanced steadily in the last twenty years such that the synthesis of artificial photo-electron transfer devices is now occurring. Even so, our understanding of the natural process of photosynthesis is far from complete. Magnetic resonance has played a crucial role in establishing the current understanding of the primary events of photosynthesis. EPR, ENDOR, RYDMR and MARY experiments on radicals, radical pairs, and triplets have provided much of the background for connecting the x-ray structure provided by Michel, Deisenhofer and Huber to the function of the various components of the bacterial reaction center. Previously, we have shown that the triplet of Rp. viridis is highly localized on one half of the special pair whereas the triplet of Rb. sphaeroides is highly delocalized such that the triplet of the special pair has approximate C2 symmetry.