Addition of NAD to pre reduced neutral red solutions did
The published electrochemical experiments that previously linked reduced NR with NAD+ ITF 2357 used a high potential drop (2000 mV) to reduce the NR without controlling the working electrode potential with a potentiostat (Jeon et al., 2012; Park and Zeikus, 1999). In addition, the solutions may have not been pre-reduced before adding NAD+. Because neutral red is known to undergo multiple redox reactions, cyclic voltammetry was performed with a large potential window (−1200 to +500 mVAg/AgCl) to determine which species may have been responsible for NAD+ reduction. While performing the scans on a phosphate buffered neutral red solution, two cathodic peaks with corresponding anodic peaks were observed (Fig. 1A). The first peak is the most commonly discussed reversible peak, which had a midpoint potential of −504 mVAg/AgCl and a cathodic peak voltage (Vpc) of −566 mVAg/AgCl. This measured midpoint potential is within 9 mV of the published value for neutral red at pH 6.0 ( Fig. S1). The second peak had a more positive midpoint potential of −346 mVAg/AgCl, but with a much larger overpotential, demonstrated by the Vpc of −909 mVAg/AgCl. This second reduction peak was noted by Halliday and Matthews (1983), but has received little attention in the recent literature. Constant polarization on the cathodic side of both peaks (−650 mVAg/AgCl and −950 mVAg/AgCl) yielded two solutions with different optical absorbance spectra ( Fig. 1B). The two molecules that give rise to these spectra are referred to in this work as NRH2 and NR−950, respectively.