BIOELECTROCHEMICAL CHARACTERIZATION OF NIFD MUTANTS FROM AZOTOBACTER VINELANDII

Nitrogenases are complex metalloenzymes that catalyze the biological reduction of atmospheric dinitrogen to ammonia, a fundamental process in the global nitrogen cycle. Their activity relies on sophisticated metalloclusters embedded within the MoFe protein, particularly the FeMo cofactor, whose protein environment is largely provided by the NifD subunit and which serves as the catalytic site for substrate reduction. The structural complexity of this cofactor and its dependence on multiple auxiliary proteins for biosynthesis and maturation impose significant challenges for heterologous expression and biotechnological applications. In contrast, industrial ammonia production remains largely dependent on the Haber–Bosch process, which operates under harsh conditions, consumes substantial amounts of energy, and contributes significantly to global CO₂ emissions. In this work, we investigate the bioelectrochemical behavior of NifD mutants from Azotobacter vinelandii and evaluate the structural and functional consequences of specific amino acid substitutions. Site-directed mutagenesis was performed using Gibson Assembly to generate variants designed to probe the roles of selected residues in FeMo cofactor stabilization. Wild-type and mutant NifD proteins were heterologously expressed in Escherichia coli, purified by nickel-affinity chromatography, and subsequently characterized using complementary bioelectrochemical and spectroscopic approaches. Protein-film electrochemistry was employed to enable direct electron transfer between the protein and the electrode surface, bypassing the physiological ATP-dependent electron-delivery pathway. Preliminary electrochemical measurements revealed enhanced cathodic currents in the presence of nitrite, consistent with electrocatalytic nitrite reduction.

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.