NADH chemistry ancillary to the oscillatory peroxidase±oxidase (PO) reaction has been reexamined. Previously, (NAD) 2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD) 2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD Keywords: horseradish peroxidase catalysis; methylene blue; NAD anomers; NAD dimer; rate constants.Peroxidases are widely distributed floral, fungal and animal heme-containing enzymes. Across the phyla, this class of enzymes participates in oxidations and reductions essential in anti-infectious defenses, hormonal signaling, and in a plants' formation and maintenance of cell walls. In mammals, peroxidases are also implicated in the pathology of inflammation. Horseradish peroxidase has found wide-spread use in a multitude of analytical assays and biosensors [1]. Due to its wide-range of catalytic activity and ready availability, HRP serves as the archetypal peroxidase in investigations of mechanisms of biochemical redox reactions. Even more importantly, pyridine nucleotides are the most common coenzymes in plant and animal cellular metabolism. NADH (Fig. 1) is one of a few substrates that activates both oxidase and peroxidase cycles of peroxidases. In stimulated neutrophils, pyridine coenzymes serve as the source of electrons shuttled by NADH oxidase through the plasma membrane to oxygen, in formation of superoxide radicals and activation of antimicrobial defenses, the chlorinating system of myeloperoxidase among them. The main target of this work is an assessment of the role of (NAD) 2 (Fig. 1) in reaction pathways operating in the horseradish peroxidase-catalyzed oxidation of NADH. A secondary goal concerns establishing the roles of other b-NADH-derived species in the PO reaction. Explorations of reduced nicotinamide adenine dinucleotide (NADH) reaction pathways are notoriously difficult. The difficulty lies in the wide range of transformations and instability of intermediates that can form. It is manifest in a large number of model compounds synthesized to restrict possible transformations. We select the HRP-NADH-O 2 oscillator as, on one hand, a more stringent framework for mechanistic evaluation of the relevant chemistry of NADH, and on the other hand, a medium kinetically enforcing a hierarchy of reaction pathways [2]. The HRP-NADH-O 2 reaction is the most thoroughly studied in the group of peroxidase oscillators [3]. The abstract models of this oscillator emulate its observable PO dynamics with general fidelity. Chemically realistic models, however, have been found lacking in simulating several features of the oscillator [4]. Our current work increases the number of reactions demonstrated to occur in the PO oscillator.In aqueous solutions of moderate acidity, the main transformations of nicotinamide adenine dinucleotide are acidcatalyzed autooxidation, anomerization, and hydration...

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In this review, we have attempted to retain the overall organizational structure of the previous review (

Inherent variance due to oscillations in the peroxidase-oxidase (PO) reaction was studied using principal component analysis (PCA). The substrates were oxygen and reduced nicotinamide adenine dinucleotide (NADH). Horseradish peroxidase (HRP) catalyzed the reaction. The concentration of a cofactor, methylene blue (MB), was varied, and 2,4-dichlorophenol was kept constant. Increase in the NADH influx was used to change the reaction dynamics from periodic to chaotic. The reaction space was abstracted to the most significant, mutually independent, pairs of absorption and kinetic basis vectors (principal components). Typically, two significant principal components were extracted from the periodic time series and three from the chaotic data. The PCA models accounted for 70-97% of experimental variance. The greatest fraction of the total variance was accounted for in experiments exhibiting periodic dynamics and less than 25 nM MB. More MB induced an increased contribution of NADH to the PO oscillator variance, as did increased NADH influx. A simulated absorption time series, computed from a mass-action model of the chemistry, was analyzed by PCA as well. The comparison of simulation with experiment indicates that the chemical model renders the time series for HRP oxidation forms with fidelity, but incompletely represents NADH chemistry and other salient processes underlying the observed dynamics.

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