Study of Nitric Oxide Chemistry/Biochemistry in S. Cerevisiae

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Nitric oxide (NO) is made in mammalian cells for a variety of physiological reasons. For example, it can serve as a vascular mediator, a cytotoxic/cytostatic species and a neurotransmitter. The biological chemistry associated with many of the actions of NO is, for the most part, extrapolated from purely chemical and/or in vitro systems. Thus far, determining the intimate chemical details responsible for the biological activity of NO has been difficult due to the limitations of in vitro or in vivo mammalian systems. However, the yeast Saccharomyces cerevisiae offers an ideal experimental system in which NO chemistry can be delineated in a whole cell environment. Due to the fact that S. cerevisiae grows and survives under anaerobic or aerobic conditions, the effect of dioxygen on the chemical interaction of NO with specific proteins can be examined. The ease of genetic manipulation of yeast allows the ability to control intracellular conditions in predictable and important ways. Thus, the effects of NO under a variety of cellular conditions can be examined. Moreover, since many of the protein relationships have been fully elucidated, alteration of protein activity by NO can be monitored by examining an overall effect on yeast cell biology. This project involves a systematic examination of the chemical and biochemical interaction of NO with two yeast metal metabolism proteins, Ace1 (a copper-responsive transcription factor) and Fre1 (a membrane spanning metal reductase). These proteins were chosen for study since they represent two important classes of proteins which are known interact with NO; metal-thiolate proteins and heme proteins. The intimate details of the NO-protein interactions will also be examined using chemical model systems and purified proteins. In this way, the cellular biochemistry of NO can be validated.