title: Turnover-dependent inactivation of the nitrogenase MoFe-protein at high pH.
abstract: Proton uptake accompanies the reduction of all known substrates by nitrogenase. As a consequence, a 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 to be problematic in view of the observation reported by Pham and Burgess [(1993) Biochemistry 32, 13725-13731] that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that the 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 a normal, albeit low, level of 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 that of the 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 formation of a tight complex with the 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 nitrogenase catalysis.
authors.affiliation: Division of Chemistry and Chemical Engineering 114-96, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Kun-Yun
authors.initials: KY
authors.lastname: Yang
authors.forename: Chad A
authors.initials: CA
authors.lastname: Haynes
authors.forename: Thomas
authors.initials: T
authors.lastname: Spatzal
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Rees
authors.forename: James B
authors.initials: JB
authors.lastname: Howard
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000737:chemistry; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D009591:Nitrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate; D009591:Nitrogenase