Activity of Inducible Nitric Oxide Synthase Requires Bound Calcium at Either Site I or Site ill of Calmodulin
Brownlow, Kaleb C.
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Nitric oxide (-NO) is a physiologically important molecule with broad cellular functions from relaxing endothelial cells to acting as a cell-cell signaling molecule. Nitric oxide synthase enzyme with both the reductase (possesses F AD and FMN) and the heme domain (possesses a protoporphyrin IX type heme) contained on the same polypeptide. Additionally, NOS requires 02 and tetrahydrobiopterin, and is a calmodulin (CaM)-dependent enzyme. Binding of CaM to NOS is required for efficient electron transfer between the domains. Previously, our laboratory demonstrated that the absence of calcium (Ca2+) bound to site I of CaM resulted in a complete loss of neuronal NOS (nNOS) activity, while the absence of Ca2+ at site III supported over 85% of nNOS activity [Stevens-Truss, et al. 1997]. By comparison the absence of Ca2+ at site I has no effect on the activity of inducible NOS (iNOS) [Stevens-Truss & MarIetta, manuscript in preparation]. This study was conducted to analyze if the absence of Ca2+ bound to site III affected activity of iNOS. Our results show that in the presence of the site III CaM activity of iNOS is 33% of its maximum. We further examined what affect a CaM that lacks Ca2+ at both sites I and III would have on iNOS activity. This CaM also only supported 33% of iNOS activity. These results were surprising to us, as we expected that in the presence of the site III CaM iNOS to be fully active, while no activity in the presence of the sites I & III CaM. We proposethat CaM interacts with iNOS in a different orientation than with nNOS. Understanding the differences between these CaM interactions and NOS could lead to novel ways of selectively inhibiting NOS isoforms.