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1. The properties of a 5-aminolaevulinic acid synthetase-deficient mutant of Escherichia coli Ki2Ymel grown aerobically or anaerobically in the presence or absence of 5-aminolaevulinic acid are described. I n the absence of 5-aminolaevulinic acid no detectable cytochromes are produced, respiration rates are lowered, and the concentration of menaquinone in the cell is increased. The absence of 5-aminolaevulinic acid from the growth medium has no apparent effect on the Mg2+. dependent ATPase activity or on the concentrations of ubiquinone and flavoprotein(s) in the cell.2. Two methods for the reconstitution of respiration by cells grown aerobically or anaerobically in the absence of 5-aminolaevulinic acid are described. With intact cells, reconstitution is achieved in 3 h at 37 "C and is dependent on the presence of 5-aminolaevulinic acid and a fermentable carbon source. With cell-free extracts, respiration can be reconstituted by the addition of haematin in 15-20 min a t 37 "C provided that ATP is also present.3. It is concluded that the reconstitution of respiration by both methods is achieved in the absence of protein synthesis, indicating that the apoproteins of cytochromes, involved in electron transport with oxygen as terminal electron acceptor, are synthesized and incorporated into the membrane in the absence of haem synthesis. 4.From an examination of the cytochromes produced during the reconstitution experiments, it is suggested that two membrane-bound electron transport systems using oxygen as terminal electron acceptor can co-exist in E . coli. The first involving cytochrome b,,, with cytochrome o as terminal oxidase is the system normally present under aerobic growth conditions, and the second is induced under conditions when the concentrations of NADH and ADP in the cell are likely to be high and involves cytochrome b668 with cytochrome a, as the terminal oxidase.Escherichia coli forms a variety of electron transport systems depending upon the conditions chosen for growth (for reviews see [l-31). Under conditions of vigorous aeration the membrane-bound carriers include ubiquinone and cytochromes b,,,, bSBB and o [4-61, whereas under conditions of anaerobic growth the major membrane-bound electron carriers are menaquinone and cytochromes b,,, bSSB and a2 together with low concentrations of cytochrome a, [6--81. Two soluble c-type cytochromes, c548 and are also produced under anaerobic growth conditions [S]; cytochrome ~5 5 0 is associated with formate-hydrogen lyase activity [9] and cytochrome c548 is only present in low concentrations and its role is unknown. Under conditions of poor aeration, Enzymes. Mga++-dependent ATPase (EC 3.6.1.3) ; hexokinase (EC 2.7.1.1); catalase (EC 1.11.1.6); 6-aminolaevulinate synthetaee (EC 6.3.-.-).Nomenclature. Nonemclature of E. wli genes is that of A. L. Taylor (1970) Bacterial. Rev. 34, 155-176. or in the late log-phase of growth, a complex mixture of all these membrane-bound electron carriers is observed [S] (and unpublished observations). Under anaerobic growt...

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.

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