A study of nitric oxide synthase from the bacterium Geobacillus stearothermophilus, and its implications for mammalian nitric oxide synthase
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Nitric oxide synthase (NOS) generates NO via a sequential two-step reaction requiring two molecules of oxygen [L-arginine (L-Arg) → N-hydroxy-L-arginine (NOHA) → L-citrulline + NO]. Each step of the reaction follows a distinct mechanism defined by each substrate (L-Arg or NOHA) bound to the heme active site. 5,6,7,8-Tetrahydrobiopterin (H4B) is a cofactor of the reaction. The reaction rate of NOS from a thermophilic bacterium, Geobacillus stearothermophilus (gsNOS) is relatively slow compared to other NOSs. Hence, gsNOS provides a unique model for studying the mechanistic differences between the two steps of the NOS reaction in greater detail.;In Chapter 2 we used equilibrium resonance Raman spectroscopy (RRS) to characterize ferric, ferrous and ferric-NO bound gsNOS under different substrate and cofactor conditions. In Chapter 3 we studied the ferrous-CO adduct of gsNOS by RRS and found the first strong evidence that the two substrates have distinct influences on both the proximal and distal sides of the heme in the active site. In Chapter 4, the application of optical stopped-flow spectroscopy to study the gsNOS reaction led us to conclude that NOHA facilitates formation of a six-coordinate water-bound ferric derivative, unique to gsNOS, after decay of the primary stable oxy-intermediate in NOS, presumably due to its active site enclosing effect (described in this chapter) as well as its proximal electron-enriching effect (elaborated on in Chapter 3). The same study indicates that the rate-determining step in both steps of the NOS reaction is the weakening of the Fe-O bond after the formation of the primary oxygen intermediate. Finally in Chapter 5, a continuous flow RRS study of the reaction between ferrous gsNOS (under different substrate and cofactor conditions) and oxygen helped us identify the primary oxy-intermediate of gsNOS to be a ferric-superoxide complex. It also allowed us to confirm observations in other NOSs of the interaction of L-Arg with the ferric-superoxide complex to promote O-O bond scission, which leads to the first step of the NOS reaction. In addition, this study showed that NOHA's effect on the ferric-superoxide complex of gsNOS is distinct from that of L-Arg.