Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli

Abstract

The respiratory NADH dehydrogenase of E. coli was further amplified in vivo by genetic methods. The enzyme, a single polypeptide of MW 47,200 of known amino acid sequence, constitutes 10-15% of the total protein in the amplified membranes. In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH: ubiquinone-1 oxidoreductase activity of .apprx. 1100-1200 U mg-1 at 30.degree. C, pH 7.5. The purified enzyme contains phospholipid which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (wt/vol) cholate at low ionic strength. Under these conditions the enzyme is extensively aggregated (apparent MW > 106). This procedure yielded enzyme with a specific activity of 980 U mg-1, similar to the value observed in the membrane. This preparation contained < 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme. Neutron activation analysis of purified enzyme demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu and Fe. The enzyme polypeptide, prepared completely free of phospholipid, FAD and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, was reactivated. The only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system were the enzyme polypeptide, FAD and phospholipid. The stereospecificity of hydride transfer from the dihydronicotinamide ring of NADH catalyzed by the enzyme both in purified form and in wild-type membranes was shown to be 4-pro-S (B). This is the same as that of the mitochondrial respiratory NADH dehydrogenase and of a group of reduced pyridine nucleotide:disulfide oxidoreductase flavoproteins which includes glutathione reductase. The stereospecificity is different from that of several other activities involved in electron transport processes or quinone reduction, which were under consideration as being evolutionarily related (e.g., NADH:-cytochrme b5 reductase).