Arginine 56 mutation in the β subunit of nitrile hydratase: importance of hydrogen bonding to the non-heme iron center

Abstract

Arginine 56 in the β subunit (βArg56) of the iron-containing nitrile hydratase (NHase), one of the strongly conserved residues within the NHase family, is known to form hydrogen bonds to the sulfinyl (–SO 2H) and sulfenyl (–SOH) groups of the post-translationally modified cysteine residues in the catalytic center. βArg56 was substituted by tyrosine, glutamate or lysine, respectively, and the respective mutant enzymes generated by reconstitution were characterized. The βR56K mutant complex exhibited about 1% of the enzymatic activity of native NHase, while the others were totally inactive. The kinetic analysis of the βR56K mutant complex exhibited a drastic decrease in turnover number and decreases in kinetic constants for substrate and inhibitors as compared to the native NHase. Changes in UV–visible absorption and light-induced Fourier transform infrared difference spectra suggest that βArg56 is involved in the positioning of the –SO 2H and –SOH groups of the modified Cys residues in the catalytic center so as to fine tune the electronic state of the iron center suitable for catalysis. Thus, βArg56 is essential for catalysis.