Bruce A. Haddock

Electron transport in aerobically grown Paracoccus denitrificans: Kinetic characterization of the membrane-bound cytochromes and the stoichiometry of respiration-driven proton translocation

John and Whatley [l] have reviewed the information so far available about the functional organization of the membrane-bound components responsible for electron transport dependent ATP synthesis in aerobically grown Paracoccus denitnficans and drew attention to the similarities that exist between these components and those of the inner mitochondrial membrane. The object of the present work was to extend these studies and to characterize further the following properties of the plasma membrane of l? denitrificans: (a) the cytochrome components identifiable by low-temperature difference spectroscopy; (b) the fast oxidation-reduction kinetics of the membrane-bound cytochromes; and (c) the stoichiometry of respiration-driven proton translocation during the oxidation of different added substrates.

The variable cytochrome content of Paracoccus Denitrificans grown aerobically under different conditions

Paracoccus denitrificans possesses a remarkable number of mitochondrial-like features including the cytochrome composition of its aerobic electron transport chain [ 1,2]. A lively controversy exists, however, as to whether the number of potential sites of energy conservation operating in heterotrophically-grown aerobic cultures of PC. denittificans is 3, as in mitochondria (e.g. [3-6]), or 2, as in bacteria like Escherichia coli (e.g. [7-l 01). A solution to this problem has been suggested, based upon changes in the relative activities of two terminal cytochrome oxidases, the mitochondrial-like cytochrome aa or the E. co&like cytochrome o, that would accept reducing equivalents from different donors in the electron transport chain [ 51. Both of these oxidases have been identified in membranes derived from aerobically grown PC. denittificans using reduced + CO minus reduced difference spectra [ 11-131 although, to date, evidence for cytochrome o functioning as a terminal oxidase under these growth conditions is lacking [4,13]. In support of this idea [5], we present here spectral and potentiometric evidence to demonstrate that PC. denitrificans can, following alterations to the aerobic growth conditions, synthesize either cytochrome aa or cytochrome o separately as the predominant terminal oxidase and, in addition, that

Immunochemical analysis of the membrane-bound hydrogenase of Escherichia coli

The membrane-bound hydrogenase [EC 1.12.-.-l ofEscherichia coli is involved in the energy-conserving oxidation of hydrogen [l-4] via fumarate reductase [EC 1.3.99.11 and also in the formate hydrogenlyase pathway which converts formate to COZ and Hz [5]. E. coli hydrogenase from aerobically grown cells has been isolated and characterised [6] and the enzyme from anaerobically grown cells has been partially characterised [7]. Antibodies specific for Bacillus subtilis membrane-bound succinate dehydrogenase [EC 1.3.99.11 have been raised from activity-stained precipitin arcs located after analysis of crude fractions by crossed immunoelectrophoresis using antisera raised to detergent-solubilised membranes [8]. We have used a similar approach to prepare antibodies specific for E. coli hydrogenase and its use has enabled the subunit MW of the enzyme from anaerobically grown cells to be determined. We also report the isolation and immunological characterisation of two new E. coli mutants which specifically lack hydrogenase activity.

Characterization of the membrane‐bound nitrate reductase activity of aerobically grown chlorate‐sensitive mutants of escherichia coli K12

Wild-type strains of Escherichia coli can grow in the presence of chlorate aerobically but under anaerobic conditions growth is inhibited. This observation has been explained on the assumption that under anaerobic conditions, chlorate, an analogue of nitrate, induces nitrate reductase (EC 1.7.99.4) and is converted by the enzyme to the toxic compound chlorite with the result that cell growth ceases: aerobic growth in the presence of chlorate is allowed since under these conditions nitrate reductase activity is repressed [ 11. Recently mutants have been isolated which show a chlorate-sensitive phenotype when grown under aerobic conditions in the presence of a fermentable carbon source [2] and a detailed biochemical characterization of one of these mutants, strain 72, has indicated that the primary genetic lesion occurs in the biosynthetic pathway for ubiquinone8 [3]. In addition, it was noticed that strain 72, when grown aerobically in the presence of nitrate, produced significantly higher activities of reduced benzylviologen-dependent nitrate reductase than an equivalent culture of the parent strain similarly grown, however this activity represented only 2-10% of that found in cultures of either strain 72 or the wild-type grown anaerobically in the presence ofnitrate [2,3]. The nitrate reductase activity found in aerobically-grown strain 72 was destroyed

Dibromothymoquinone: An inhibitor of aerobic electron transport at the level of ubiquinone in Escherichia coli

Dibromothymoquinone (DBMIB)** inhibits photosynthetic [l] and mitochondrial [2] electron transport by acting as an antagonist for benzoquinone function in both systems. Since there are few sitespecific inhibit0r.s of electron transport in Escherichia coli, other than KCN [3], we have investigated the effect of DBMIB on the growth and respiratory chain of aerobic cultures of this bacterium. In this paper we demonstrate that DBMIB inhibits: a) growth of the organism on a non-fermentatable carbon source, b) NADH, D-lactate and D,L a-glycerophosphate oxidase activities together with NADHand D-lactate DCPIP reductase activities of membrane particles, and c) NADH-dependent cytochrome b reduction. In addition we show that in the presence of membrane particles, substrate, and KCN, DBMIB catalyzes a rapid oxygen uptake activity that is apparently cytochrome independent but can result in the partial oxidation of the b-type cytochromes. We conclude that DBMIB acts as an antagonist of ubiquinone function in the aerobic respiratory chain of E. coli on the substrate side of the cytochromes [4] but, because the compound can undergo rapid reduction and autooxidation under certain conditions, its general application as an inhibitor of electron transport is limited.

MICROCALORIMETRIC MEASUREMENTS OF HEAT EVOLUTION AND THEIR CORRELATION WITH OXYGEN UPTAKE IN ESCHERICHIA COLI WITH GENOTYPICALLY- AND PHENOTYPICALLY-MODIFIED ELECTRON TRANSPORT CHAINS

The use of microcalorimetry for investigation of microbial metabolism is of considerable and growing interest [l] . Recently, Long et al. [2] described the heat evolution associated with active transport of nonmetabolisable compounds by Escherichia coli. Previously, a direct correlation between heat evolution and oxygen uptake during growth of a variety of micro-organisms (including E. cd) had been’reported [3]. This proportionality was independent of growth rate, but a dependence on growth substrate and type of organism was proposed. We have used a sensitive microcalorimeter to measure heat evolution following addition of fermentable and non-fermentable substrates to various strains of E. coli, possessing modified electron-transport chains. The results demonstrate a close correlation between heat evolution and the rate of oxygen uptake.

Assembly of functional b-type cytochromes in membranes from a 5-aminolaevulinic acid-requiring mutant of Escherichia coli

The investigation of the properties of the respiratory chain components in E. colt has been greatly aided by the analysis of various mutants [1-3] . The formation of functional enzymes may be studied through investigation of the effects of genetic lesions on the synthesis of apoproteins and prosthetic groups, and their assembly. E. colt is capable of synthesizing a number of distinct cytochromes [4,5] but the isolation of mutants deficient in their ability to produce specific cytochromes has not been reported. However cytochrome biosynthesis has been investigated using mutants requiring 5-aminolaevulinic acid, a presursor of haem biosynthesis, for non-fermentative growth [6-8] . Functional cytochromes are synthesized by these mutants during growth in the presence of 5-aminolaevulinic acid, but when grown in the absence of this compound no cytochromes are spectrally detectable and oxidase activities are extremely low [7]. When membranes derived from cells grown aerobically in the absence of 5-aminolaevulinic acid are incubated with haematin and ATP, NADH oxidase activity can be reconstituted in vitro. This reconstitution was shown to be independent of protein synthesis [7] implying that apocytochromes were pre-assembled in the membrane. Since these membranes were incubated with haematin (haem b), only b-type cytochromes were reconstituted. The role of ATP in the reconstitution of NADH oxidase activity is unclear but it has been shown that hydrolysis of the ATP by an enzyme other

Phosphate transport and the stoicheiometry of respiratory driven proton translocation in Escherichia coli

Abstract The stoicheiometry of respiratory driven proton translocation associated with the oxidation of endogenous substrates has been measured in an organic phosphate auxotrophic mutant of Escherichia coli . The results obtained indicate that movements of inorganic phosphate do not result in an experimental underestimation of the observed H+/site ratio in E. coli , as has previously been suggested for mitochondria.

On the role of lipoic acid as a cofactor for oxidative phosphorylation in Escherichia coli

Abstract Using an auxotrophic mutant of Escherichia coli and the technique of quenching of atebrin fluorescence by membrane particles it has been shown that lipoic acid is not required for either respiration or ATP-driven proton tranlocation.