Nitric Oxide synthases produce NO as an important molecular signal or as a defensive cytoxin in organisms ranging from archaea to mammals. Eukaryotic NOS isoforms are large modular enzymes; NO production by signaling enzymes is controlled by calcium regulation of electron flux from NADPH to the oxygenase active site via the biosensor calmodulin. Controlled production of NO is central to many physiological feedback systems. The FMN binding domain delivers electrons as it shuttles between the NADPH dehydrogenase and oxygenase catalytic centers in the enzyme. Electrons enter from the NADPH binding site and are donated at the oxygenase site. Experiments will study the formation of the state in which electrons enter the oxygenase site. The mechanism of the electron shuttle will be described in detail. The project will concentrate on nNOS, a Ca+2/CaM controlled signal generator, and iNOS, which is Ca+2 insensitive, to describe the shuttle mechanism and its activation by calmodulin in detail. The experimental plan emphasizes techniques accessible to advanced undergraduates. The proposal demonstrates intellectual merit by providing new paradigms for NOS function and control and novel tests of central concepts. Underlying concepts include the tethered shuttle model, design of input and output state constructs, and new kinetics models.
Broader Impacts As a RUI project, the research program has an integral training component including undergraduate student participation in preparative and experimental work. At least four undergraduate students each summer will participate, and additional students work on the project during the academic year. They will get advanced training and experience in preparative and experimental biochemistry and molecular genetics. Students will learn how to design a project, how to plan and carry out a line of experimental work, and how to analyze and evaluate data. Computationally advanced students will learn the basics of simulation. Students will all have the opportunity to attend a regional or national scientific conference and present work. Preparation of presentations is important because it provides experience in writing, organization, and production of meaningful figures. As a result, about twenty undergraduates with diverse backgrounds will be introduced to science as a living intellectual endeavor. Some of them will be recruited into science and allied professions as a career, but all of them will be better equipped to make decisions about their own career choices and, as citizens, about issues on local and national scale.
. J.C.Salerno, PI) This project was aimed at understanding the mechanism and control of endothelial nitroc oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), two related enzymes that generate nitric oxide (NO) as a molecular signal. NO is a neurotransmitter, and is an important signal in the regulation of vascular tone (hence blood pressure), cardiac function, angiogenesis (the development of new blood vessels), insulin secretion, and many other biological processes. A third isoform, inducible nitric oxide synthase (iNOS) is involved in immune response has the same catalytic machinery but is controlled primarily at the level of expression (how much of the enzyme is made). We used advanced methods to measure eNOS and nNOS binding and release from calmodulin (CaM), the primary activator, and to examine the effects of phosphorylation (addition of a negatively charged phosphate group) by regulating kinases on CaM binding. Protein kinase C (PKC), an inhibitor of NO production, but not the activating kinases PKA and AKT, prevented CaM binding. A paper describing these findings, Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin, was published in FEBS Journal. We also reported fluorescence experiments that revealed a series of obligatory conformations in the nitric oxide synthase catalytic cycle characterized by their fluorescence lifetimes (how long the excited state that gives rise to fluorescence persists after excitation by light; this is a function of the environment of the fluorescent group). This work has been recently published in FEBS Journal (FMN fluorescence in iNOS constructs reveals a series of conformational states involved in the reductase catalytic cycle.) This work provided important information that verified our ‘tethered shuttle’ hypothesis for the mechanism of electron delivery by nitric oxide synthases and enzymes with related reductase components such as P450 reductase. We then showed that nitric oxide production by both eNOS and nNOS is turned on by calmodulin binding through changes that make the enzymes more flexible. Briefly, because of their very different fluorescence lifetimes, we can now distinguish three types of conformations: input conformations in which the FMN cofactor of the enzyme is reduced (given electrons) by NADPH via the FAD cofactor, output conformations in which FMN reduces heme, and a series of ‘open’ states in which FMN is not associated with other cofactors. Calmodulin binding speeds up the transitions between these states, leading to changes in the distribution of states within the conformational manifold because not all transitions are equally affected. CaM activation of NOS favors the output and open states at the expense of the majority input state. The first paper describing this work was published in FEBS Letters (Calmodulin activates neuronal nitric oxide synthase by enabling transitions between conformational states). This provides a paradigm for NOS activation, and in addition gives us a powerful probe to study the effects of other regulators on the enzymes. As part of this effort, the PI’s group refined its proposed mechanism for the reductase catalytic cycle of NOS and related enzymes, producing a global mathemetical (King-Altman) model for NOS and P450 reductase type enzymes. This model accounts for essentially all available steady-state enzyme kinetics data, including data that we obtained specifically to test the model. In a significant breakthrough, we recognized that the major control element of eNOS contains a binding site for MAP kinases, an important group of signaling enzymes. This developed when eNOS diverged from nNOS via paired frame shift mutations that produced a variable region. Wild type eNOS, but not nNOS and not eNOS deletion mutants, bind MAP kinases; this binding was partially inhibited by calmodulin. These observations strongly suggest a direct role for MAPK in regulation of NOS with implications for signaling pathways including angiogenesis and control of vascular tone. Our initial paper (Map kinases bind endothelial nitric oxide synthase Carol A. Chrestensen, Jonathan L. McMurry & John C. Salerno, FEBS Open Access) recently appeared in FEBS Open Access, the new online journal of the Federation of European Biochemistry Societies. Our paper describing regulation of eNOS by phosphorylation by the MAP kinase ERK has just been accepted by Biosciences Reports, the open access journal of the Biochemical Society. Ten Kennesaw undergraduates participated in the project. Three have been accepted by graduate or professional programs, and two are working in the biotechnology industry. Two minority students won significant university, regional and national awards. Ezibobiara Umejiaego was named student of the year by the Kennesaw State biology program, out of 1200 students. He won two awards from the regional NSF-LSAMP program, an award from the national BEYA program, and was named a national Merck Scholar. Vladimir Moricette won a regional NSF-LASMP award and an award from the national BEYA program. Normal 0 false false false EN-US JA X-NONE
