Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH
| Autor: | Thomas Spatzal, James B. Howard, Kun-Yun Yang, Douglas C. Rees, Chad A. Haynes |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Molybdoferredoxin Conformational change Time Factors Crystallography X-Ray Biochemistry Article chemistry.chemical_compound Adenosine Triphosphate ATP hydrolysis Nitrogenase chemistry.chemical_classification Azotobacter vinelandii biology Chemistry Hydrolysis Substrate (chemistry) Hydrogen-Ion Concentration biology.organism_classification Enzyme assay Enzyme biology.protein Biophysics Adenosine triphosphate |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi4014769 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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