L. F. Oltmann

The existence of an alternative electron-transfer pathway to the periplasmic nitrite reductase (cytochrome cd1) in Paracoccus denitrificans

Exposure of aerobically-grown wild-type cells of Paracoccus denitrificans to a decreased aeration caused parallel increases in both PMS/ascorbate and succinate-linked activities of nitrite reductase. By contrast, the expression of the succinate-linked activity was considerably delayed in an insertion mutant specifically lacking the periplasmic 15 kDa cytochrome c-550. In this case the observed activity followed very closely the content of a 40 kDa cytochrome c. A subcellular fraction enriched in a haemoprotein of a similar apparent molecular weight showed the activity of cytochrome c peroxidase and was able to restore the antimycin-sensitive electron transport from membrane vesicles to nitrite reductase. It is concluded that P. denitrificans possesses an alternative nitrite-reducing pathway involving the 40 kDa cytochrome c instead of cytochrome c-550. This pathway branches from the respiratory chain after the cytochrome bc1 segment.

The Membrane-bound Cytochromes of an Aerobically Grown, Extremely Thermophilic Bacterium, PS3: Characterization by Spectral Deconvolution Coupled with Potentiometric Analysis

SUMMARY: Cytochromes of the a, b and c types in membranes from the thermophilic bacterium PS3 have been characterized with respect to their spectral, potentiometric and ligand-binding properties. The integrated approach used (a) conventional potentiometric analysis with non-linear least-square analysis, (b) incorporation of the potentiometric procedures into a spectral decomposition protocol at 298 and 77 K, (c) fourth-order finite difference and fourth derivative analysis, and (d) photodissociation studies of the CO-liganded components. Either one c-type cytochrome with a split α-band (547, 552 nm at 77 K) or two cytochromes c with similar redox potentials lapprox. +230 mV) were found, together with at least three b-type cytochromes (554, 557 and 561 nm at 77 K). On the basis of its high redox potential (+104mV), cytochrome b 551 was tentatively equated with the o-type oxidase of this organism. A c-type cytochrome that formed a photodissociable complex with CO was also detected. The α-band of the previously purified cytochrome c oxidase was resolved into two components, having spectral and potentiometric properties remarkably similar to those of the analogous mitochondrial oxidase.

Linkage of formate hydrogenlyase with anaerobic respiration in Proteus mirabilis

The linkage between the enzyme system catalysing formate hydrogenlyase and reductases involved in anaerobic respiration in intact cells of anaerobically grown Proteus mirabilis was studied. Reduction of nitrate and fumarate by molecular hydrogen or formate was possible under all growth conditions; reduction of tetrathionate and thiosulphate occurred only in cells harvested at late growth phase from a pH-regulated batch culture and not in cells harvested at early growth phase or in cells grown in pH-auxostat culture. Under all conditions, cells possessed the enzyme tetrathionate reductase. We conclude that linkage between tetrathionate reductase (catalysing also reduction of thiosulphate) and the formate hydrogenlyase chain is dependent on growth conditions. During reduction of high-potential oxidants such as fumarate, tetrathionate (when possible) or the artificial electron acceptor methylene blue by formate, there was no simultaneous H2 evolution due to the formate hydrogenlyase reaction. H2 production started only after complete reduction of methylene blue or fumarate, in the case of methylene blue after a lag phase without gas production. In preparations with a low fumarate reduction activity this was accompanied by an acceleration in CO2 production. During reduction of thiosulphate (a low-potential oxidant) or of tetrathionate in the presence of benzyl viologen (a low-potential mediator) by formate, H2 was evolved simultaneously. From this we conclude that formate hydrogenlyase is regulated by a factor that responds to the redox state of any electron acceptor couple present such that lyase activity is blocked when the acceptor couple is oxidised to too great an extent.

Reduction of oxygen by hydrogen in cells of anaerobically grown Proteus mirabilis

Abstract Anaerobically grown cells of the enterobacteria Proteus mirabilis, Escherichia coli, Salmonella typhimurium and Enterobacter cloacae catalyse the reduction of oxygen by hydrogen (The Knallgas reaction). We have studied this reaction in detail in P. mirabilis. Oxygen at concentrations above approx. 20 μM inactivates the catalytic pathway for this reaction in a reversible way. The site of inactivation is located in the part of the pathway shared with the reduction of fumarate by hydrogen, possibly hydrogenase. Oxygen exerts its effect via the oxidation state of an unknown component in the bacteria, such that electron transfer from H2 is blocked when this component is oxidised. We suggest that this component is identical to the regulating factor controlling hydrogen production via the formate-hydrogenlyase reaction (Krab, K., Oltmann, L.F. and Stouthamer, A.H. (1982) Biochim. Biophys. Acta 679, 51–59).

The functional localization of cytochromes b in the respiratory chain of anaerobically grown Proteus mirabilis

The functional localization of the cytochromes b found in anaerobically grown Proteus mirabilis was investigated. From light absorption spectra, scanned during uninhibited and HQNO-inhibited electron transport to various electron acceptors, it was concluded that all cytochromes b function between two HQNO inhibition sites, or more probably in a Q- or b-cycle.

Purification and partial characterization of the membrane-bound haem-containing proteins from Acinetobacter calcoaceticus LMD 79.41

Summary: Cytoplasmic membranes of Acinetobacter calcoaceticus cells, cultured under acetate-limiting conditions, contain cytochrome b554 and a cytochrome o-containing oxidase. Both have been purified to homogeneity and characterized. Cytochrome b554 is a monomeric protein (molecular mass 70 kDa) with a midpoint potential of +100 mV (in the membrane it has most probably a value of +50 mV). The cytochrome o-containing oxidase seems to be an αβγδ protein since the molecular mass of the native protein was estimated to be 150 kDa and the molecular masses of the subunits, determined by SDS-polyacrylamide-gel electrophoresis, are 55, 41, 33 and 17 kDa. Redox spectroscopy of the purified complex shows the presence of a cytochrome b555/563 having a midpoint potential of approximately +160 mV (both in purified form and in the membrane). CO difference spectroscopy shows the presence of a second b-type cytochrome, viz. cytochrome, o. Cytoplasmic membranes of A. calcoaceticus cells grown under oxygen-limiting conditions also show the presence of the cytochrome b554 and the cytochrome o-containing oxidase. In addition a protein has been solubilized with the spectral characteristics of a cytochrome d-containing oxidase. The cytochrome o- and d-containing oxidases appear to be similar to those reported for Escherichia coli and Proteus mirabilis. On the other hand, cytochrome b554 has no counterpart in these organisms since the cytochrome b556 described for E. coli is quite dissimilar.

The b-type cytochromes of aerobically grown Proteus mirabilis in electron transport to oxygen

Electron transport in a bacterium like Proteus mirabilis is still poorly understood. Depending on the growth condition different electron transport chains are synthesized, catalysing electron transport to different terminal electron acceptors. Here, we report about the function of the band c-type cytochromes in electron transport to oxygen in cytoplasmic membranes from aerobically grown cells. Spectrophotometrically four cytochrome components can be distinguished (absorption maxima at 563, 556, 557 and 549 nm), whereas a further characterization by coupled potentiometric analysis and spectrum deconvolution of a set 77K-spectra of cytoplasmic membranes poised at different redox-potentials (see Van Wielink et al., 1982) reveal that the 557 nm component arises from two b-type cytochromes, one with a midpoint potential o f about + 50 mV and one with a midpoint potential of about 110 mV. The 563 nm component and the 556 nm component possibly arise from one double-peaked b-type cytochrome (midpoint potential approximately + 140 mV). The 549 nm component (midpoint potential approximately +75 mV and probably a c-type cytochrome) contributes for only 2 5 ~ to the 540-580 nm absorbance band, so it is not very likely that this cytochrome is an obligatory link in electron transport from N A D H to oxygen. Analyses of 77K-spectra o f cytoplasmic membranes, sampled during steady-state electron transport from N A D H to oxygen, reveal that the 563, 556 and 549 nm components and probably the 557 nm ( + 50 mV) component feel a similar redox-potential (approximately + 180 mV). Since this redox-potential is not really altered in the presence of a concentration of HQNO, that inhibits electron transport from N A D H to oxygen for 90%, it is concluded that there is an H Q N O inhibition site before and behind these cytochromes. Furthermore, it is found that part of the 557 nm component (propably the 110 mV component) is slowly reduced and reoxidized, so it is not very likely that this b-type cytochrome has a function in electron transport from N A D H to oxygen. The observations above are consistent with an electron transport chain, in which cyt. b-557 ( + 50 mV), cyt. b-563 ( + 140 mV) and cyt. b-556 ( + 140 mV) or alternatively cyt. b-557 ( + 5 0 mV) and cyt. b-563 556 ( + 140 mV) are localized in the main electron transport chain to oxygen, between two H Q N O inhibition sites. Two cytochromes, cyt. e-549 ( + 7 5 mV) and cyt. b-556 ( 1 1 0 mV), do not seem to form part of the main electron transport chain.