ABSTRACT
Direct electrochemical studies, utilizing two voltammetric methods-square-wave voltammetry (SWV) and cyclic voltammetry (CV)-have been performed on recombinant forms of the flavin domain of spinach assimilatory nitrate reductase in the presence of NAD+ analogs. The reduction potentials (E degrees ') of the flavin domains have been determined at an edge pyrolytic graphite electrode utilizing MgCl2 as a redox-inactive promoter. Under identical experimental conditions (pH 7.0, 25 degrees C), the two-electron reduction potential for the FAD/FADH2 couple has been determined to be -274 and -257 mV by SWV and CV, respectively. In contrast, the reduction potentials of free FAD have been determined to be -234 and -227 mV by SWV and CV, respectively. The reduction potentials of the complex formed between the FAD prosthetic group in the recombinant flavin domain and various NAD+ analogs have been determined to be as follows: NAD+ (E degrees ' = -192 mV), 5'-ADP ribose (E degrees ' = -199 mV), ADP (E degrees ' = -154 mV), AMP (E degrees ' = -196 mV), adenosine (E degrees ' = -192 mV), adenine (E degrees ' = -220 mV), and NMN (E degrees ' = -208 mV). In contrast to these positive shifts in reduction potential, nicotinamide (E degrees ' = -268 mV) had very little effect on the reduction potential of this flavin complex. Moreover, addition of NAD+ to the FAD prosthetic group in a variety of mutant forms of the recombinant flavin domain resulted in positive shifts in the reduction potential of the complex, although the magnitude of the shifts varied from a minimum of 6 mV obtained for the C240A mutant to a maximum of 79 mV obtained for the C62S mutant. These results represent the first extensive application of direct electrochemistry to examine the redox properties of assimilatory nitrate reductase and indicate that complex formation with NAD+, or various NAD+ analogs, results in a positive shift in the flavin reduction potential, with the magnitude of the shift correlating well with the efficiency of the inhibitor.
