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The enzymatic mechanisms and energetics of nitrogenase-catalyzed photoproduction of hydrogen from organic C-compounds by purple bacteria are discussed in respect to the question of which of the following three enzymes or enzyme systems are rate limiting for H2-production: (a) the nitrogenase-complex; (b) the enzymes and electron transport proteins involved in hydrogen transfer from the organic substrate(s) to nitrogenase; and (c) the system of photosynthetic ATP-regeneration. Calculations of maximum in vivo rates of photosynthetic ATPregeneration (gATP-values derived from growth rates), and of ATP-consumption by nitrogenase- catalyzed H2-production, and comparison of these rates with the specific activities of the enzymes of hydrogen or electron transfer from the C-substrate to the nitrogenase-complex, make it very likely that, in Rhodospirillum rubrum, nitrogenase-catalyzed H2-formation is limited by the availability of ATP and, possibly, of reducing power. In two other nonsulfur purple bacterial species (Rhodobacter capsulatus and Rhodobacter sphaeroides), H2-photoproduction is probably not energy-limited.

A first model of the three-dimensional structure of the photosynthetic reaction center of the mutant T1 (SerL 223 → Ala, ArgL 217 → His) from Rhodopseudomonas viridis, resistant toward the triazine herbicide terbutryn (2-methylthio-4-ethylamino-6-f-butylamino-5-triazine), has been developed from X-ray data measured to a resolution of 2.5 Å. The secondary quinone, QB, which in T1 binds better than in the wild type, is present in the crystals. Both substituted residues are clearly visible in the difference fourier map. The replacement of these two residues in the QB site causes only minor changes in the overall structure of the protein.

Four atrazine-resistant mutants from the purple bacterium Rhodopseudom onas viridis were isolated. Sequence analysis revealed three different mutant strains carrying mutations in the herbicide-binding pocket: i) M AV 2: L 212-Glu → Lys, ii) M AV 3: L216-Phe → Ser and iii) MAV 4 = MAV 5: L217-Arg → His, L220-Val → Leu. Except M AV 3 all Rps. viridis mutants are different from those selected by their resistance towards the closely related triazine terbutryn.

Among 4 strains of the purple bacterium Rhodospirillum rubrum only one did not contain trimethylamine N-oxide/dimethylsulfoxide reductase and was unable to grow under anaerobic dark conditions in fructose media supplemented with trimethylamine N-oxide (TMAO) or dimethylsulfoxide (DMSO ). By growing the bacteria in media treated with charcoal or supplemented with tungstate (2 mM ), TM AO/DMSO reductase activity was lowered to about 10% of the value of the untreated control. The enzyme was purified from the periplasmic protein fraction of strain S1 and was shown to contain 2 subunits (mol. wt. of 40,000 and 36,000, respectively) in a 1:1 stoichiometry. Although the affinity of the reductase to DMSO (Km - 0.67 mM) was much higher than to TMAO (Km = 24 m M ) , the catalytic activity with the latter substrate was about 10 times as high (90-110 μmol/min X mg protein) as with DMSO

Fluorescence induction characteristics have been studied in wild-type and atrazine-resistant mutant of Rliodobacter capsulatus. Fluorescence induction was found to be a useful technique to monitor the altered electron transfer in the atrazine-resistant mutants as well as in the different membrane fractions of wild-type R. capsulatus. In both cases, the proportion of the fast rise of variable fluorescence was increased indicating the enhancement of QA. I`n the mutant strain, the I50 value of triazine herbicide terbutryn was increased by 100-fold whereas the natural resistance of R. capsulatus against diuron was abolished by the mutation.

Two c-type cytochromes (a high spin cytochrome c′ and a low spin cytochrome c-553(549) with asymmetrical α-band) and a low spin cytochrome b-558 from the purple sulfur bacterium Ectothiorhodospira shaposhnikovii were purified by ion exchange chromatography and gel filtration and characterized. Cytochrome c′ has a molecular weight of 33000 (determined by sodium dodecylsulfate electrophoresis), an isoelectric point at pH 4.5 and a redox potential of +37 mV. Absorption spectra show in the oxidized state maxima at 404 nm and in the range of 635 nm, in the reduced form maxima at 426.5 nm, 549 nm and a shoulder at 435 nm. The best purity index obtained was 0.48 (A280/A426.5). Reduced cytochrome c′ reacts with carbon monoxide. Cytochrome c-553(549) has a molecular weight of 10400, an isoelectric point at pH 5.1 and a redox potential of +248 mV. The oxidized form shows the Soret-band at 410 nm. The reduced protein reveals an asymmetrical a-band at 553 nm with a shoulder at 549 nm, the a-band at 522 nm with a shoulder at 528 nm and the γ-band at 416 nm. The best purity index obtained was 0.18 (A280/A416). Roth cytochromes could be isolated from the soluble fraction as well as from Triton X-100 treated membranes. Furthermore very low amounts of cytochromes c-553 and c-552.5 could be detected in detergent treated chromatophores. Cytochrome 6-558 - obtained from cells grown in the presence of reduced sulfur compounds in the medium - seems to be soluble or only weakly bound to the membrane. It has a molecular weight of 15800. an isoelectric point at pH 4.1 and a redox potential of -210 mV. The hemoprotein shows absorption maxima at 424.5 nm. 526.5 nm and 556.5 nm in the reduced form and at 416 nm in the oxidized state. The best purity index obtained was 0.26 (A280/A42,4.5). In addition, there were hints for the occurrence of a high spin cytochrome b′. The cytochrome pattern as well as the amount of cytochromes were dependent on growth conditions.

The purple sulfur bacterium T. roseopersicina is able to grow chemoautotrophically in the dark under micro- to semiaerobic conditions. Under the latter cell yield and growth rate are considerably reduced. During chemo- and phototrophic growth reduced sulfur compounds are metabolized by the same pathway, i.e. oxidized to sulfate via adenylylsulfate. APS reductase (EC 1.8.99.2), ADP sulfurylase (EC 2.7.7.5) and ATP sulfurylase (EC 2.7.7.4) could be shown in all the strains tested, whereas only strain BBS contained an AMP independent sulfite-oxidizing activity under photo- as well as under chemotrophic conditions. Not only ADP but also ATP sulfurylase perform a dissimilatory function proven by their high specific activities. In contrast to the enzyme of T. roseopersicina strain 6311 APS reductases from strains M1 and BBS are strictly membrane-bound. The enzyme from strain M1 was solubilized, enriched and characterized. While the KM values of purified APS reductase remain unaffected, specific activities of APS reductase, ATP and ADP sulfurylase are increased substantially under chemotrophic growth conditions.

A series of pigments of the bacteriopheophytin a spectral type have been isolated from ageing cultures of Rhodopseudomonas sphaeroides strain R26. These pigments are formed in varying amounts, and can be most readily analyzed in vivo by their absorption in the 530 nm spectral range. They are enriched in sedimenting cells, but their formation is not affected by light. By chromatographic comparison with authentic pigments and chemical correlation, the following pigments have been identified: bacteriopheophytin a, bacteriopheophorbide a (which is the predominant product), pyrobacteriopheophorbide a and a fourth, very polar bacteriopheophytin a-type product of unknown structure. The major portion of these newly formed pigments is present in the cells in a state, in which the near-infrared absorption band is shifted to longer wavelengths. As shown by low temperature fluorescence spectroscopy, these forms are very similar to bacteriopheophorbide a aggregates.

The mutant strains A l a+ and Y 5 of Rhodopseudomonas capsulata are defective in formation of the light-harvesting complexes B800-850 and B870, respectively, but synthesize the large bacteriochlorophyll-binding polypeptides of the respectively complexes, shown by amino acid composition, N- or C-terminal sequence and immunoblotting. The stable assembly of the light-harvesting complexes in the membrane seems to be dependent from the coordinated synthesis of all pigment and protein components.

Photsynthetic membranes of Rhodopseudomonas virids and Ectothiorhodospira halochloris were treated with proteinase K. The photochemical activity (light minus dark difference spectra) were compared to the polypeptide composition (SDS-polyacrylamide gel analysis). In E. halo­chloris, difference bands appear at 806 (+), 838 (+) and 854 nm (-) . All three decrease in intensity upon incubation with proteinase K., but this decrease is much slower than the proteolysis of both the reaction center and antenna related polypeptides. Photochemical activity remains high as long as a small part of the RC and two lower molecular weight polypeptides M* (22.0 kDa) and B* (15.3 kDa) are present. The M subunit is the most stable polypeptide in the RC of Rp. viridis too, and the photochemical activity is related to the remainder of this and to the one newly formed polypeptide (15.3 kDa), but doesn’t show the typical absorption shift of the antenna (B 800/1020 → B 800/960). The results are discussed quantitatively and compared to those obtained from Bchl α containing organisms.

Fluorescence detected ODMR measurements in zero field on the triplet states of light harvest­ing (LH) pigments in chromatophores from Rhodopseudomonas sphaeroides R 26.1 and Rhodo­pseudom onas capsulata A1a+ are reported. In both cases at least two distinct triplet states in the antenna system with zfs values of D between 209 and 217 and E between 56 and 67 x 10-4 cm-1 are observed.

Fluorescence detected ODMR measurements in zero field on the triplet state of the reaction center in whole cells of Rhodopseudomonas viridis, in reaction center solution and in reaction center crystals are reported. In solution and in the crystal a sign reversal of the F-ODMR signals is observed by variation of the wavelength of detection. The signs of the signals are interpreted with the Vredenberg-Duysens relation.

Sulfonyl urea-herbicides (sulfometuron methyl, chlorsulfuron) strongly inhibit phototrophic growth of the bacterium Rhodospirillum rubrum in the absence of ʟ-valine as a feedback inhibitor of the enzyme acetohydroxy acid synthase. The latter enzyme was shown to be highly sensitive to the sulfonyl urea and, to a much lesser extent, to an imidazolinone herbicide (Arsenal®).