NITROGENASE REACTIVITY - EFFECTS OF PH ON SUBSTRATE REDUCTION AND CO INHIBITION

Molybdenum nitrogenase is composed of two separately purified proteins designated the iron protein (Fe protein) and the molybdenum-iron prot ein (MoFe protein), with the latter containing the substrate reduction site which is a metal cluster designated the iron-molybdenum cofactor (FeMo cofactor). In addition to its physiological substrates H+ and N 2, nitrogenase reduces a number of nonphysiological substrates (e.g. C 2H2 and N3-) and interacts with a number of similar molecules (e.g. CH 3NC and CO) that serve as specific inhibitors. Despite their great div ersity, all substrates are reduced by multiples of two electrons and r equire equivalent numbers of electrons and protons. Although the elect ron donor to a substrate is believed to be FeMo cofactor, the nature o f the proton donor is unknown and might be different for different sub strates. Here we report a three-component buffer assay system that eli minates variables of buffer type, ionic strength, and ATP and reductan t availability and that is compatible with the nitrogenase system in t he pH range 5.0-9.8. Preincubated studies and studies of the effects o f pH on H-2 evolution under Ar, H-2 evolution under N2, H-2 evolution under CO, and C2H2 reduction show that there is a group with a pK of c a. 6.3 that must be deprotonated for substrate reduction to occur and that there is a group with a pK of ca. 9.0 that must be protonated for substrate reduction to occur. The pK of the 9.0 group is shifted 0.5 pH unit in the acid direction by both CO and C2H2 but not by N2, while the pK of the 6.3 group is shifted 0.4 pH unit in the acid direction by C2H2 but not by CO or N2. Thus, CO appears to inhibit H-2 evolution by native nitrogenase at high pH. These substrate effects strongly su ggest that the groups being titrated are at or near the active, FeMo c ofactor, site of the enzyme.