Characterization of azido-NAD+ to assess its potential as a two-dimensional infrared probe of enzyme dynamics

Abstract

Enzyme active-site dynamics at femtosecond to picosecond time scales are of great biochemical importance, but remain relatively unexplored due to the lack of appropriate analytical methods. Two-dimensional infrared (2D IR) spectroscopy is one of the few methods that can examine chemical biological motions at this time scale, but all the IR probes used so far were specific to a few unique enzymes. The lack of IR probes of broader specificity is a major limitation to further 2D IR studies of enzyme dynamics. Here we describe the synthesis of a general IR probe for nicotinamide-dependent enzymes. This azido analog of the ubiquitous cofactor nicotinamide adenine dinucleotide is found to be stable and bind to several dehydrogenases with dissociation constants similar to that for the native cofactor. The infrared absorption spectra of this probe bound to several enzymes indicate that it has significant potential as a 2D IR probe to investigate femtosecond dynamics of nicotinamide-dependent enzymes.

Scheme 1

Synthetic scheme for the production of azo-NAD⁺ from azopyridine and NAD⁺ (−).

Figure 1

Normalized UV absorption spectra of azo-NAD⁺ (−), azopyridine (…), and NAD⁺ (− −).

Figure 2

IR absorption spectra of azopyridine (upper panel) and azo-NAD⁺ (lower panel).

Figure 3

UV absorption spectra showing the conversion of azo-NAD⁺ to azo-NADH.

Figure 4

IR absorption spectra of azo NAD⁺ in solution (−) and bound to FDH (−).

Figure 5

IR absorption spectrum of azo-NAD⁺ bound to FDH with azide anion.