Probing the Role of a Conserved Phenylalanine in the Active Site of Thiocyanate Dehydrogenase

Abstract

Copper-containing enzymes catalyze a broad spectrum of redox reactions. Thiocyanate dehydrogenase (TcDH) from Thioalkalivibrio paradoxus Arh1 enables the bacterium to use thiocyanate as a unique source of energy and nitrogen. Oxidation of thiocyanate takes place in the trinuclear copper center of TcDH with peculiar organization. Despite the TcDH crystal structure being established, a role of some residues in the enzyme active site has yet to be obscured. F436 residue is located in the enzyme active site and conserved among a number of TcDH homologs, however, its role in the copper center formation or the catalytic process is still not clear. To address this question, a mutant form of the enzyme with F436Q substitution (TcDHF436Q) was obtained, biochemically characterized, and its crystal structure was determined. The TcDHF436Q had an unaltered protein fold but did not possess enzymatic activity, whereas it contained all three copper ions, according to ICP-MS data. The structural data showed that the F436Q substitution resulted in a disturbance of hydrophobic interactions within the active site crucial for a correct transition between open/closed forms of the enzyme–substrate channel. Thus, we demonstrated that F436 does not participate in copper ion binding, but rather possesses a structural role in the TcDH active site.