INVESTIGATION OF CO BINDING AND RELEASE FROM MO-NITROGENASE DURING CATALYTIC TURNOVER

During enzymatic turnover in the presence of CO, Mo-nitrogenase has be en shown to generate two different EPR signals termed lo-CO (P-CO = 0. 08 atm) and hi-CO (P-CO = 0.5 atm). When the formation of hi-CO is mon itored under the conditions of very low electron flux, a 2 min lag is observed prior to the initial detection of the signal followed by a ne ar-linear rate of formation during which the S = 3/2 cofactor signal e xhibits similar decay kinetics. Increasing the electron flux produces a significant increase in the rate of both the formation of hi-CO and the decay of the S = 3/2 cofactor. These results are interpreted in te rms of a state of the enzyme (redox or structural) generated only duri ng turnover which is needed to initially bind CO to the cofactor. Unde r high electron flux conditions, new EPR inflections are observed at g = 5.78, 5.15 and g = 1.95, 1.81 and tentatively assigned to S = 3/2 a nd 1/2 states of the GO-bound cofactor and 1 equiv of oxidized P clust er, respectively. Sudden removal of CO from the environment results in the slow decay (>10 min) of both the hi-CO signal and CO inhibition o f acetylene reduction activity. The use of ethylene glycol to quench e nzymatic activity strongly inhibits the decay of hi-CO (in the presenc e of CO) and the subsequent decay of lo-CO (after removal of CO) but d oes not prevent the reversible interconversion hi-CO <-> lo-CO + CO.