EVIDENCE FOR MULTIPLE STEPS IN THE PRE-STEADY-STATE ELECTRON-TRANSFERREACTION OF NITROGENASE FROM AZOTOBACTER-VINELANDII

The effect of the NaCl concentration and the reaction temperature on t he MgATP-dependent pre-steady-state electron transfer reaction (from t he Fe protein to the MoFe protein) of nitrogenase from Azotobacter vin elandii was studied by stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy. Besides lowering the reaction temperature, also the addition of NaCl decreased the observed rate constant and the amplitu de of the absorbance increase (at 430 nm) which accompanies pre-steady -state electron transfer. The diminished absorbance increase observed at 5 degrees C (without NaCl) can be explained by assuming reversible electron transfer, which was revealed by rapid-freeze EPR experiments that indicated an incomplete reduction of the FeMo cofactor. This was not the case with the salt-induced decrease of the amplitude of the st opped-flow signal: the observed absorbance amplitude of the electron t ransfer reaction predicted only 35% reduction of the MoFe protein, whe reas rapid-freeze EPR showed 80% reduction of the FeMo cofactor. In th e presence of salt, the kinetics of the reduction of the FeMo cofactor showed a lag period which was not observed in the absorbance changes. It is proposed that the pre-steady-state electron transfer reaction i s not a single reaction but consists of two steps: electron transfer f rom the Fe protein to a still unidentified site on the MoFe protein, f ollowed by the reduction of the FeMo cofactor. The consequences of our finding that the pre-steady-state FeMo cofactor reduction does not co rrelate with the amplitude and kinetics of the pre-steady-state absorb ance increase will be discussed with respect to the present model of t he kinetic cycle of nitrogenase.