4ND8

Av Nitrogenase MoFe Protein High pH Form

Summary for 4ND8

• Entry DOI: 10.2210/pdb4nd8/pdb
• Related: 1M1N
• Descriptor: Nitrogenase molybdenum-iron protein alpha chain, Nitrogenase molybdenum-iron protein beta chain, 3-HYDROXY-3-CARBOXY-ADIPIC ACID, ... (7 entities in total)
• Functional Keywords: high ph, oxidoreductase, nitrogen fixation, nifh
• Biological source: Azotobacter vinelandii; More
• Total number of polymer chains: 4
• Total formula weight: 233237.20

Authors

Yang, K.-Y.,Haynes, C.A.,Spatzal, T.,Rees, D.C.,Howard, J.B. (deposition date: 2013-10-25, release date: 2014-01-15, Last modification date: 2023-09-20)

Primary citation

Yang, K.Y.,Haynes, C.A.,Spatzal, T.,Rees, D.C.,Howard, J.B.
Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH.
Biochemistry, 53:333-343, 2014

PubMed 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.

PubMed: 24392967
DOI: 10.1021/bi4014769

Experimental method

X-RAY DIFFRACTION (2 Å)