Josef Wachtveitl

Initial characterization of site-directed mutants of tyrosine M210 in the reaction centre of Rhodobacter sphaeroides.

A description of the properties of site‐directed mutants of the reaction centre (RC) of Rhodobacter sphaeroides is presented. The residue tyrosine M210 (YM210) has been changed to phenylalanine (FM210) and leucine (LM210). Both mutants grew photoheterotrophically under conditions of high light but only the FM210 mutant grew under low light. Photobleaching spectra of chromatophores isolated from these mutants showed that the amount of functional RC was comparable to wild‐type and that the spectral features were essentially unchanged. Shifts were observed in the absorption spectra in regions attributable to all the chromophores. An increase in intensity and a 3 nm red shift (from 803 to 806 nm) was observed in the Qy band of the monomer bacteriochlorophylls. A new extinction coefficient for the RC was determined at 806 nm (332 +/‐ 15 mM‐1 cm‐1). Linear dichroism (LD) spectra showed that there was no significant large scale change in the angles of the individual pigments relative to the C2 axis of symmetry. Cytochrome turnover assays were performed on isolated RC and light harvesting I complex (LH I)‐RC (photosynthetic units, PSU) preparations. A turnover number of 120 cyt RC‐1 s‐1 was calculated for both the mutants while wild‐type had a turnover number of 228 cyt RC‐1 s‐1. The cytochrome c2‐mediated re‐reduction kinetics of P+ were comparable to those observed in the wild‐type. The half‐time of charge recombination within the RC increased in the mutants to the wild‐type (100 ms in the wild type, and 150 and 200 ms in FM210 and LM210, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Herbicide biosensor based on photobleacing of the reaction centre of Rhodobacter sphaeroides

Abstract A fast and simple measuring device for the detection of photosystem-II herbicides is presented. Herbicides are monitored by absorption changes at 860 nm after photobleacing for 2 s. This system can be easily miniaturized and consists of three components, sample cell, light source and detector for absorption measurements, and a computer data processing unit. Detection ranges and limits were determined for several herbicides. Plans for further miniaturization are discussed.

Identification of a hydrogen bond in the Phe M197→Tyr mutant reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides by X‐ray crystallography and FTIR spectroscopy1

In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X‐ray structure analysis of the Phe M197→Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 Å resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875–12886, 1993] between the Tyr M197 hydroxy group and one of the 2a‐acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the π‐conjugated 9‐keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.

13C MAS NMR evidence for structural similarity of L162YL mutant and Rhodobacter sphaeroides R26 RC, despite widely different cytochrome c2-mediated re-reduction kinetics of the oxidized primary donor

CPMAS NMR data collected from L162YL mutant [4′-13C]Tyr-enriched Rhodobacter sphaeroides RCs reveal that Tyr L162 is in a slightly heterogeneous and probably rigid section of the protein complex. The differences in chemical shifts of the individual components relative to those of the [4′-13C]Tyr Rhodobacter sphaeroides R26 response are 0.2 ppm or less. This is small compared to the total dispersion of [4′-13C] isotropic shifts, ∼ 5 ppm, which measures the shift range due to variations in the microscopic environment between the various tyrosines in the protein complex. The structural changes in the mutant with respect to Rhodobacter sphaeroides R26, as probed by the labels, are thus minimal on the scale of the NMR. This suggests that the dramatic decrease of re-reduction rate of the oxidized primary donor P upon mutation (Farchaus et al., Biochemistry 32 (1993) 10885–10893) cannot be attributed to significant structural changes in the protein. Hence the NMR is in line with the current view that the decrease of the re-reduction rate in the mutant originates from slow reorientation of the docked cytochrome.

The puf B,A,L,M Genes are Not Sufficient to Restore the Photosynthetic Plus Phenotype to a puf L,M,X Deletion Strain

The purple non-sulfur bacterium Rhodobacter sphaeroides has the capacity to thrive as an anaerobic phototroph. The ability to convert light energy into chemical energy within the cell is driven by a photosynthetic unit consisting of two light-harvesting antennae complexes and a reaction center (RC). The light-harvesting complexes have been labelled LHI and LHII based on their respective single (875 nm) and double (800–850 nm)infrared absorption maxima. The RC responsible for the primary light driven electron transfer is composed of three protein subunits H, M and L and contains 4 molecules of bacteriochlorophyll a (Bchl a), 2 molecules of bacteriopheophytin a, one non-heme iron and two molecules of ubi-quinone. Two of the Bchl a molecules are monomeric and are referred to as the voyeur Bchl while the other two form a dimer, or so-called special pair (1). The RC special pair undergoes a reversible photobleaching that can be measured at 860 nm or 600 nm. These two maxima have been assigned to the Qy and Qx transition bands of the special pair, respectively (2).

Light Harvesting, Energy Transfer and Photoprotection in the Fucoxanthin-Chlorophyll Proteins of Cyclotella meneghiniana

The excitation energy transfer and the protective role of diadinoxanthin and diatoxanthin in two different fucoxanthin-chlorophyll-proteins have been investigated using femtosecond transient absorption spectroscopy.

On the Role of Tyrosine L162 in the Re-Reduction of the Reaction Center Special Pair by Cytochrome C2

The crystallization and X-ray structure analysis of the reaction centers (RC) from Rhodopseudomonas viridis (1) and Rhodobacter sphaeroides (2, 3) has provided detailed information about the chromophores and their surrounding protein environment. These data allow one to identify amino acids which could serve as direct mediators and/or structural components vital for light-driven electron transport.

Primary Reaction of Sensory Rhodopsin II Mutant D75N

The primary reaction of the sensory rhodopsin II mutant D75N has been investigated using femtosecond transient absorption spectroscopy. A reaction mechanism taking into account all observations including the slower photoresponse has been worked out.

The role of tyrosine M210 in the initial charge separation in the reaction center of Rhodobacter sphaeroides

Site-directed mutants of tyrosine M210 (YM210) in the reaction center (RC) of Rhodobacter sphaeroides have been constructed and characterized to determine the effect of the changes on both the structure of the RC and its electron transfer kinetics. YM210 has been replaced by phenylalanine (FM210) and leucine (LM210). Both mutants are able to grow photosynthetically under high light but under low light the LM210 mutant is photosynthetic minus. Both mutant strains contain equal amounts of photobleachable RC in the intracytoplasmic membrane (normalized to total bacteriochlorophyll) as compared to wild type. Photobleaching spectra of mutant membranes are basically indistinguishable from wild type. Absorption spectra of purified mutant RCs also are basically the same as the wild type with small changes observed in the position and intensity of the Qy transition of the monomer bacteriochlorophylls. Picosecond kinetic analysis shows that both the lifetime of the excited state of the primary electron donor (P*) and the rise time of the first reduction step are longer in both mutants. These processes occur in 3.5 ps in the wild type RC while the corresponding time constants in the mutants are 16 ± 6 ps in FM210 and 22 ± 8 ps in LM210.

Coherent Effects in the Carbonyl Containing Carotenoid Fucoxanthin

Coherent effects in the isolated carbonyl containing carotenoid fucoxanthin in various solvents and fucoxanthin within the fucoxanthin-chlorophyll protein were investigated using femtosecond transient absorption spectroscopy.