Electron transport to nitrogenase in Azotobacter chroococcum. Purification and some properties of NADH dehydrogenase.

Abstract

NADH dehydrogenase from Azotobacter chroococcum was solubilised and purified to yield a single band on disc‐gel electrophoresis. The molecular weight of the enzyme was approximately 450000 and it contained one subunit of molecular weight approximately 16000. The enzyme transferred electrons from NADH to triphenyl tetrazolium chloride, benzyl viologen, potassium ferricyanide, NAD or horse‐heart cytochrome c, and from NADPH to NAD or potassium ferricyanide. The pH optimum for benzyl viologen or triphenyl tetrazolium chloride was between pH 9 and 10, that for potassium ferricyanide or acetyl‐pyridine‐NAD between pH 6.5 and 8.0. There were two pH optima with cytochrome c: at 8.0 and 9.5. The rate of reduction of tetrazolium salt or benzyl viologen by purified NADH dehydrogenase at pH 7.5 was not proportional to protein concentration; activity at low concentrations was restored by adding either azotoflavin, azotobacter ferredoxin or nitrogenase. NADH‐cytochrome‐c‐reductase activity was more heat‐sensitive and more susceptible to inhibition by p‐hydroxymercuribenzoate than were NADH‐ferricyanide or NADH benzyl viologen reductases. NADH‐ferricyanide reductase was inhibited by organic solvents and NADH benzyl viologen or tetrazolium reductase was inhibited by Cu2+ ions. These results are discussed in terms of separate active centres for the reductions. NADH dehydrogenase coupled to nitrogenase to reduce acetylene in the presence of ATP, benzyl viologen and NADH.