The adenosine triphosphate requirement for nitrogen fixation in cell-free extracts of Clostridium pasteurianum

Abstract

1. The requirements for the decomposition of acetyl phosphate in extracts of Clostridium pasteurianum have been shown to be the same as those for N₂ fixation with H₂ as the electron donor-H₂, ATP, ferredoxin and Mg²⁺. The decomposition of acetyl phosphate (ATP) under H₂ is not caused by an activation of enzymes by H₂, nor by an ATP-dependent uptake of substrate amounts of H₂. 2. Removal of ATP from extracts by charcoal treatment allowed N₂ fixation from pyruvate to proceed appreciably when fixation from H₂ was almost completely inhibited. Addition of increasing amounts of ATP to extracts freed of ATP by Sephadex gel-filtration showed that a much lower concentration of ATP-supported N₂ fixation from pyruvate than from H₂. One site of ATP action was thus localized between H₂ and reduced ferredoxin. 3. A requirement for ATP in pyruvate metabolism has been established and may explain the ATP requirement for N₂ fixation from pyruvate. It appears doubtful that ATP is required at the site of N₂ reduction. 4. H₂ acted as a competitor of N₂ fixation when H₂ was the electron donor, but acetyl phosphate (ATP) consumption increased with increasing pH₂. When N₂ fixation was completely inhibited by either CO or N₂O, ATP consumption was only slightly decreased. Separate enzymes apparently function in ATP consumption and N₂ reduction. 5. Mg²⁺ is required for ATP consumption as well as for ATP generation from acetyl phosphate by acetokinase (ATP: acetate phosphotransferase, EC 2.7.2.1). 6. A scheme is presented for the action of ATP in N₂ fixation; an activated form of reduced ferredoxin produced enzymically with ATP is considered to function in N₂ fixation.