Reiner Feick

Isolation and characterization of light harvesting bacteriochlorophyll · protein complexes from Rhodopseudomonas capsulata

The isolation of two native light harvesting bacteriochlorophyl.protein complexes from Rhodopseudomonas capsulata is described. The light harvesting bacteriochlorophyll I (B 875) has been isolated from the blue-green mutant A1a+ lacking both carotenoids and light harvesting bacteriochlorophyll II. Light harvesting bacteriochlorophyll I is associated with a protein (light harvesting band 2) of 12 000 molecular weight. Light harvesting bacteriochlorophyll II complex has been isolated from the mutant Y5 lacking a reaction center and light harvesting bacteriochlorophyll I. Light harvesting bacteriochlorphyll II (B 800 + 850) together with carotenoids is associated with two polypeptides (light harvesting bands 3 and 4) having molecular weights of about 8000 and 10 000 (sodium dodecyl sulfate polyacrylamide gel electrophoresis). A third protein (light harvesting band 1) is in the purified light harvesting II fraction (mol. wt. approx. 14 000), but not associated with bacteriochlorophyll or carotenoids. The amino acid composition of the 3 antenna pigment II proteins is given. The polarity of these proteins was found to be 48%. From the amino acid composition the following molecular weights were calculated band 1: 17 350, band 3: 13 350 and band 4: 10 500.

Subpicosecond emission studies of bacterial reaction centers

The spontaneous emission of reaction centers from native and mutated Rhodobacter sphaeroides and from wild type Chloroflexus aurantiacus is investigated by fluorescence up-conversion with high temporal resolution. The time constant of 0.9 ps previously observed in transient absorption experiments on wild type reaction centers of Rhodobacter sphaeroides does not appear in the emission experiment. However, all investigated reaction centers display a biexponential decay of the emission with time constants in the 2 ps to 25 ps range. The experimental results are discussed within the frame of different reaction models including a possible sample heterogeneity or a transient electron transfer to the inactive pigment branch.

Determination of free energies in reaction centers of Rb. sphaeroides

Abstract Magnetic field-dependent recombination measurements together with magnetic field-dependent triplet lifetimes (Chidsey, E.D., Takiff, L., Goldstein, R.A. and Boxer, S.G. (1985) Proc. Natl. Acad. Sci USA 82, 6850–6854) yield a free energy change ΔG( P + H − − 3 P ∗) = 0.165 eV ±0.008 at 290 K. This does not depend on whether nuclear spin relaxation in the state 3P∗ is assumed to be fast or slow compared to the lifetime of this state. This value, being (almost) temperature independent, indicates ΔG( P + H − − 3 P ∗) ⋍ ΔH( P + H − − 3 P ∗) and is consistent with ΔG( 1 P ∗ − P + H − ) and ΔH( 1 P ∗ − 3 P ∗) from previous delayed fluorescence and phosphorescence data, implying ΔG ⋍ ΔH for all combinations of these states.

Observation of acttvationless recombination in reaction centers of R. Sphaeroides: A new key to the primary electron-transfer mechanism

Abstract Activationless recombination of the primary radical pair of R. Sphaeroides in its triplet state supports the involvement of superexchange electronic coupling in the primary electron-transfer step in bacterial reaction centers.

Fluorescence emission by wild-type- and mutant-strains of Rhodopseudomonas capsulata

Absorption and fluorescence emission spectra of Rhodopseudomonas capsulata, strains 37b4 (wild type), A1a+ (blue-green mutant strain), Y5 (phototroph negative, having only B-800--850 bacteriochlorophyll-carotenoid-protein complex) at 4 K, 77 K and 300 K were measured. The fluorescence emission at 890 nm of the B-870 bacteriochlorophyll band dominates the emission of other spectral forms of the strains 37b4 and A1a+, while in strain Y5 a fluorescence emission band at 865 nm of the B-850 bacteriochlorophyll dominates. Very little fluorescence was observed at 805 nm. A linear relation between relative fluorescence intensity and the exciting light intensity was observed. The integrated fluorescence yield increased as the temperature was lowered from 300 K to 4 K. The results are discussed in the light of the arrangement of pigment molecules in the membrane and the process of energy migration within the photosynthetic apparatus.

A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing

A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.

The primary structure of two chlorosome proteins from Chloroflexus aurantiacus

The complete nucleotide sequence of two chlorosome proteins with apparent molecular weights of M r 18,000 and M r 11,000 from Chloroflexus aurantiacus have been determined. The two polypeptides were 145 and 97 amino acids long and possessed true molecular masses of 15,545 and 10,820 Da, respectively. Protein chemical sequencing was done in parallel to confirm the primary structure deduced from nucleotide sequencing. By Northern blot analysis of RNA isolated from phototrophically grown cells a transcript of 0.95 kb was detected which is the expected length for a mRNA encoding both genes.

Biochemical and Spectroscopic Properties of the Reaction Center of the Green Filamentous Bacterium, Chloroflexus aurantiacus

The reaction center of the thermophilic green bacterium Chloroflexus (Cf.) aurantiacus has a bacteriochlorophyll donor-bacteriopheophytin-quinone acceptor system which is characteristic for purple bacteria and not for Chlorobiaceae reaction centers. It is comprised of 3 BChl a-, 3 BPheo a-, 1-2 menaquinone molecules and only two protein subunits with almost identical molecular masses of approximately 35,000 Da. The RC complex possesses thermal stability. Despite the absence of the so called H-protein subunit, electron transfer steps are not impaired although the kinetics are somewhat slower than that in the three protein subunit RC of purple bacteria. Hence, the RC of Cf. aurantiacus is the smallest functional RC isolated thus far. The two polypeptides possess a moderate identity of 40% each to the L- and M proteins of Rhodobacter sphaeroides and Rhodopseudomonas viridis RC, respectively. A characteristic feature of the N-terminus of the Cf. aurantiacus RC is the extended hydrophilic region at the N-terminus. When compared to purple bacteria RC, a few functionally important amino acids are not conserved; these differences help to explain some of the spectral and kinetic features of the Cf. aurantiacus RC.

High quantum yield of charge separation in reaction centers of Chloroflexus aurantiacus

The relative quantum yield of charge separation in photosynthetic reaction centers of Chloroflexus aurantiacus and Rhodobacter sphaeroides was measured on the nanosecond timescale. Thereby the quantum yield becomes independent of a potential loss of both quinones during reaction center isolation. Based on the quantum yield of 1.02±0.04 for reaction centers of Rhodobacter sphaeroides at room temperature (Wraight, C.A. and Clayton, R.K. (1973) Biochim. Biophys. Acta 333, 246–260), the quantum yield of initial charge separation in reaction centers of Chloroflexus aurantiacus at 280 K was determined to be 1.06 ± 0.11.

Cloning and sequencing of the genes encoding the light-harvesting B806-866 polypeptides and initial studies on the transcriptional organization of puf2B, puf2A and puf2C in Chloroflexus aurantiacus

The genes encoding the α-and β-polypeptide subunits of the B806-866 membrane-bound light-harvesting complex of Chloroflexus aurantiacus have been cloned and the nucleotide sequences determined. The gene puf2A, which encodes the B806-866 α-polypeptide, began 28 bases downstream of the stop codon of puf2B, which encodes the B806-866 β gene. The gene-encoding cytochrome c-554, puf2C, was found about 250 bp downstream of puf2A. puf2A encoded a 13 amino acid extension at the C-terminus of the B806-866 α-polypeptide that was not present in the mature protein. These genes, unlike those of purple nonsulfur bacteria, did not form a contiguous operon with puf1L or puf1M, the genes encoding the L and M subunits of the photochemical reaction center. The occurrence of the two latter genes and of puf2B and puf2A in two separate operons has not been observed in purple bacteria. Under photoheterotrophic growth conditions, puf2B and puf2A were encoded on an abundant mRNA that was 0.5 kb long. Two monocistronic transcripts for puf2C were observed that had different 5′-ends. One transcript encoding all three genes was also detected. Nucleotide sequences very similar to the consensus promoter sequence of the Escherichia coli RNA polymerase σ70 subunit were found seven and eight bases upstream of the 5′-end of mRNA encoding puf2B and for one of the monocistronic mRNA encoding puf2C, respectively.