We have measured the Raman spectra of oxidized nicotinamide adenine dinucleotide, NAD+, and its reduced form, NADH, as well as a series of fragments and analogues of NAD+ and NADH. In addition, we have studied the effects of pH as well as deuteration of the exchangeable protons on the Raman spectra of these molecules. In comparing the positions and intensities of the peaks in the fragment and analogue spectra with those of NADH and NAD+, we find that it is useful to consider these large molecules as consisting of component parts, namely, adenine, two ribose groups, two phosphate groups, and nicotinamide, for the purpose of assigning their spectral features. The Raman bands of NADH and NAD+ are found generally to arise from molecular motions in one or another of these molecular moieties, although some peaks are not quite so easily identified in this way. This type of assignment is the first step in a detailed understanding of the Raman spectra of NAD+ and NADH. This is needed to understand the binding properties of NADH and NAD+ acting as coenzymes with the NAD-linked dehydrogenases as deduced recently by using Raman spectroscopy.

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the binding nature of an aromatic aldehyde to the catalytic site of liver alcohol dehydrogenase from horse (LADH) using preresonance Raman spectroscopy. The compound p-(dimethylamino)benzaldehyde (DABA) is converted to the corresponding alcohol in the presence of nicotinamide adenine dinucleotide (NADH) and a catalytic amount of enzyme at neutral pH. A stable ternary complex of LADH/NADH/DABA can be formed if enzyme and coenzyme are in excess at high pH [Jagodzinski, P. W., Funk, G. F., & Peticolas, W. L. (1982) Biochemistry 21, 2193-2202]. We have obtained the preresonance Raman spectrum of bound DABA by subtracting the contribution of the binary complex of LADH/NADH from the spectrum of this stable ternary complex. In order to understand the normal mode patterns of DABA, four isotopically labeled DABA derivatives were synthesized and their Raman spectra, in solution and in the ternary complex, were measured. Three of these compounds contain substitutions in the functionally important aldehyde moiety: (i) In one such substitution, the aldehydic hydrogen atom was replaced by a deuterium; (ii) in another, this hydrogen atom was replaced by deuterium, and the aldehydic carbon atom was replaced by 13C; and (iii) in the third derivative, only the carbon atom was replaced by 13C. The fourth derivative has had the two hydrogen atoms at the 3- and 5-positions of the DABA ring replaced by deuterium atoms. We find that many of the spectral modes are fairly extended, involving both stretching and bending motions of the entire molecule, although a few modes are quite localized. We find that the normal mode structure of DABA changes considerably when it binds to LADH/NADH. As a model for the bound DABA, we have examined the zinc complexes of DABA (and all four isotopically labeled samples) in anhydrous diethyl ether and methylene chloride. A striking correspondence between the Raman spectra of the enzyme-bound DABA and DABA-Zn complexes in solution is found, which extends to all the isotopically labeled derivatives. This suggests that one of the major roles of LADH in the binding of DABA is to provide a divalent zinc ion to form a first-sphere Lewis acid complex. The data also suggest other interactions between enzyme-bound DABA with its protein surroundings and with the coenzyme NADH are quite minor. An estimate of the carbonyl bond character of bound DABA had been made on the basis of the response of Raman bands to isotopic labeling and on trends observed in spectra of DABA in solvents of various polarities.(ABSTRACT TRUNCATED AT 400 WORDS)

We report the first Raman spectra of reduced nicotinamide adenine dinucleotide (NADH) when bound to an enzymatic active site, that of liver alcohol dehydrogenase (LADH). This was obtained by subtracting the Raman spectrum of LADH from that of the binary LADH/NADH complex. There are significant changes in the spectrum of bound NADH as compared to that in solution. The data indicate that both the nicotinamide moiety and the adenine moiety are involved in the binding. At least one of the two NH2 moieties of NADH also participates.

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