1. Assay conditions are described for the ATP-dependent, uncoupler-sensitive, energy-linked reduction of NAD + by succinate, dl -α-glycerophosphate or d -lactate in membranes from aerobically grown Escherichia coli . 2. The reaction may be demonstrated in electron-transport particles (ET particles) from cells grown in glycerol, but not in depleted particles washed in low-ionic-strength buffer, or in ET particles from cells grown in glucose. 3. The latter two classes of particles have low specific activities of ATPase (adenosine triphosphatase), succinate dehydrogenase, dl -α-glycerophosphate dehydrogenase and d -lactate dehydrogenase relative to undepleted ET particles from cells grown in glycerol. 4. Reconstitution of energy-linked NAD + reduction in particles from cells grown in glucose was done by: ( a ) addition of the high-speed supernatant fraction from sonicates of the same cells; ( b ) addition of a protein fraction, precipitated by (NH 4 ) 2 SO 4 from this supernatant, or ( c ) addition of an (NH 4 ) 2 SO 4 -precipitated fraction from the low-ionic-strength wash of particles from cells grown in glycerol. 5. The use of (NH 4 ) 2 SO 4 -precipitated fractions from ATPase- or succinate dehydrogenase-deficient mutants grown in glycerol in the above reconstitution indicated that failure to demonstrate the reaction in particles from cells grown in glucose was a result of inadequate activities of appropriate dehydrogenases, rather than of ATPase. 6. Energy-linked NAD + reduction could be demonstrated in particles from a ubiquinone-deficient mutant only after restoration of NADH oxidase activity by adding ubiquinone-1. 7. The measured rate of the energy-linked reaction in particles from a haem-deficient mutant, however, was not stimulated after the ATP- and haematin-dependent acquisition of functional cytochromes. 8. Results are interpreted as evidence of the ubiquinone-dependent, but cytochrome-independent, nature of the site I region of the respiratory chain in E. coli .

Measurements were made of electron-transport-dependent quenching of atebrin fluorescence in particles prepared from mutants of Escherichia coli unable to synthesize either ubiquinone or haem. Such quenching was either absent (in haem-deficient particles) or decreased (in ubiquinone-deficient particles), but was restored under conditions used to reconstitute oxidase activities. It is concluded that the reconstitution of oxidase activity, in both cases, is associated with the formation of a functional, proton-translocating, respiratory chain.

1. The reconstitution of oxidase activity in cell-free extracts of a mutant of Escherichia coli K12Ymel, that require 5-aminolaevulinic acid for growth on non-fermentable carbon sources, is described. 2. The reconstitution is dependent on haematin or a haem extract from a prototrophic strain of E. coli , and the product of the reaction has been identified as NADH-reducible cytochrome b . 3. The requirement for haematin cannot be replaced by four other porphyrins. Coproporphyrin III does not inhibit the haematin-dependent reconstitution, mesoporphyrin IX and protoporphyrin IX apparently compete with haematin for a binding site on the cytochrome apoprotein(s) and deuteroporphyrin IX binds to cytochrome apoprotein(s) and cannot be subsequently replaced by haematin. 4. The properties of electron-transport particles from cell-free extracts of the mutant strain, grown aerobically in the presence or absence of 5-aminolaevulinic acid, are described. In the absence of 5-aminolaevulinic acid no detectable cytochromes are produced, and oxidase activities are lowered but there is no apparent effect on the activities of the NADH dehydrogenase and d -lactate dehydrogenase. 5. The reconstitution of oxidase activity by electron-transport particles from cells grown in the absence of 5-aminolaevulinic acid requires ATP and haematin, and the product of the reaction was identified as NADH-reducible cytochrome b . 6. It is concluded that the cytochrome apoproteins are synthesized and incorporated into the cytoplasmic membrane of E. coli in the absence of haem synthesis. The subsequent reconstitution of functional cytochrome(s) requires protohaem, but the nature of the side chain on the 2 and 4 positions of the porphyrin appears to be important.

Measurements were made of the stoicheiometry of respiration-driven proton translocation coupled to the oxidation of NAD(P)-linked or flavin-linked substrates in intact cells of Escherichia coli . Observed stoicheiometries (→H + /O quotient; Mitchell, 1966) were approx. 4 with l -malate as substrate and approx. 2 for succinate, d -lactate and glycerol oxidation. It is concluded that the potential number of equivalent energy-conservation sites associated with the respiratory chain is 2 in aerobically grown cells of E. coli harvested during the exponential phase of growth.