Proton uptake accompanies the reduction of all known substrates by nitrogenase. As one consequence, higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high pH approach would appear problematic in view of the observation reported by Pham and Burgess (Biochemistry 32, 13725 (1993)) that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While normal, albeit low, substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially the enzyme is reversibly inhibited for substrate reduction at pH 9.5 yet in a second, slower process the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to native MoFe-protein yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by tight complex formation with Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism based transformation that may provide a new probe of the nitrogenase catalysis.