The effects of point mutations at belt-sulfur sites on nitrogenase catalysis
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Abstract
BACKGROUND: Although the current understanding proves that dinitrogen reduction occurs at NifDK protein’s M-cluster, the exact reduction mechanism remains controversial and is under active research. Recent crystallographic studies from our laboratory have suggested an asymmetric displacement of belt sulfurs of the M-cluster by three dinitrogen species that possibly represent different reduction states of N2. The current theory proposed by my colleagues is that substrate catalysis occurs through a belt-sulfur mobilization mechanism (Hu, 2022). The three sulfur sites are situated in the center of M-clusters, and they are named S2B, S3A, and S5A. As the substrate-bound clusters rotate through the three belt-sulfur sites in the order of S3A, S2B, and S5A, stepwise substrate reduction takes place, with the S5A site serving as the exit point for reduced products.
OBJECTIVE: Our lab is interested in the intricate mechanism of enzymatic substrate reduction. With the crystallographic evidence of asymmetrical belt-sulfur displacements during substrate turnover, this research project aims to test the catalytic ability of the nitrogenase enzyme when the three belt-sulfur sites are altered by site-directed mutagenesis.
METHODS: Eight sets of Azotobacter vinelandii nifDK genes (encoding the catalytic NifDK component of nitrogenase) with various point mutations at belt-sulfur sites were each transformed into E. coli, followed by cell growth and heterologous expression of each NifDK variant. NifDK variant proteins were then isolated, purified, and analyzed. A series of analytical assays were conducted to test for the formation of NifDK components, iron contents in M-cluster, and the ability to perform substrate reduction.
RESULTS: All NifDK mutants, except YM532 and YM533, showed no acetylene reduction activity. YM532 and YM533 showed some, but significantly low acetylene reduction activity. In addition, YM533 showed further reduction from ethylene to ethane. As expected, all eight mutants are cofactor-deficient but contained P-clusters; however, the P-cluster contents of all mutants, as reflected by the presence of less iron atoms, were lower than that of the positive control.
CONCLUSIONS: Mutations to belt-sulfur sites led to completely abolished or significantly diminished substrate reduction activities of the nitrogenase enzyme while other conditions were held constant. This result confirms the critical role of belt-sulfur sites in nitrogenase catalysis.
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