Evidence for the Existence of a Fully Reduced State of Molybdoferredoxin during the Functioning of Nitrogenase, and the Order of Electron Transfer from Reduced Ferredoxin

Abstract

Abstract Molybdoferredoxin from Clostridium pasteurianum W5 was studied under conditions where the nitrogenase system was turning over and molybdoferredoxin was known to be ∼90% in the EPR-silent state. Optical spectroscopy showed that the EPR-silent molybdoferredoxin species thus formed was not the same as the EPR-silent species formed by dye oxidation. The steady state (EPR-silent) molybdoferredoxin and the EPR-positive form of molybdoferredoxin isolated from cells in the presence of dithionite were oxidized with a 1.3-fold excess thionin and the numbers of electrons transferred to thionin were calculated. It was found that the EPR-silent molybdoferredoxin species formed by incubation with azoferredoxin, MgATP, and dithionite held 4 more transferable electrons per tetramer (2 more per dimer) than EPR-positive molybdoferredoxin. Oxidized azoferredoxin could be reduced by reduced ferredoxin, whereas oxidized molybdoferredoxin could not be reduced by reduced ferredoxin unless both azoferredoxin and MgATP were present. A reduction scheme for N2 fixation is proposed.