The reconstitution of oxidase activity in membranes derived from a 5-aminolaevulinic acid-requiring mutant of Escherichia coli.

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.