PURIFICATION AND CHARACTERIZATION OF THE RESPIRATORY NITRATE REDUCTASE OF BACILLUS-LICHENIFORMIS

1. 1. Respiratory nitrate reductase of Bacillus licheniformis was extracted from the bacterial membranes by treatment with deoxycholate and purified to a homogeneous state by means of gel chromatography and anion-exchange chromatography. 2. 2. The enzyme (Mr = 193 000, s20,w = 8.6) consists of two subunits, having apparent molecular weights of 150 000 (α subunit) and 57 000 (β subunit), which are present in an equimolar ratio. It does not contain carbohydrate. Ageing of the enzyme appears to result in splitting of the polypeptide chains at specific sites followed by dissociation and reassociation of the digestion products in various combinations. 3. 3. In contrast to Klebsiella aerogenes respiratory nitrate reductase, which is isolated in a tetrameric form that can be reversibly dissociated into a monomeric form by detergents, B. licheniformis nitrate reductase, after isolation, is always present in a monomeric form. This property is related to the difference in membrane localization of the enzyme in the two organisms. 4. 4. B. licheniformis nitrate reductase contains 6.9 atoms of non-heme iron, 6.7 atoms of acid-labile sulfide and 0.93 atoms of molybdenum per molecule of enzyme. The molybdenum seems to be part of a low-molecular weight peptide (Mo-cofactor) to which it may be bound by interaction with thiol-groups. 5. 5. Antiserum against the native enzyme contains antibodies against both subunits as well as the Mo-cofactor. The Mo-cofactor does not have any antigenic determinants in common with either the α or the β subunit. Also neither subunit cross-reacts with antiserum against the other subunit. Whereas the respiratory nitrate reductases from K. aerogenes and Escherichia coli are immunologically related, the native enzyme from B. licheniformis does not show any cross-reaction with antiserum prepared against either the K. aerogenes or the E. coli enzyme. © 1979.