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Nitric Oxide Synthase

People working on this project:

Joshua Woodward
Theodor Agapie
Steve Reece


Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to •NO and citrulline. The critical biological role of •NO is now well-established, both in signal transduction and in the host response to infection (1, 2). In signal transduction it serves as a cell-to-cell signaling agent involving stimulation of the synthesis of the second messenger guanosine 3':5'-cyclic monophosphate (cGMP) in the target cell, as illustrated below.
On the other hand, the immune system appears to have harnessed the toxic properties of •NO, using this molecule to kill or inhibit the growth of invading organisms. There are three isoforms whose tissue distribution and regulation largely determine physiological action. The enzyme is a unique and complicated redox protein (3-5). Biochemical studies and sequence comparisons have shown that NOS contains two tightly bound flavin cofactors that function to deliver reducing equivalents to a bound heme. The heme is a cytochrome P-450 type heme with a cysteinyl thiolate providing a ligand to iron. The enzyme also contains a tightly bound pterin cofactor.  The overall enzymatic mechanism leading to •NO formation is under investigation. Our studies to date have involved various aspects of structure and catalysis and future investigations will continue to explore these areas. A general summary of the current projects are described below.
 
Expression and site-directed mutagenesis - We have succeeded in working out methods to express both the inducible isoform of NOS (iNOS) and the neuronal isoform (nNOS). nNOS has been expressed both in a baculovirus system (6) as well as in E.coli (7). Our first mutagenesis studies (C415H and C415A) were carried out with the baculovirus system. These mutations were made to locate the cysteine ligand to the iron-heme (6, 8). Although the mutants did not bind heme, we were able to use them to identify C415 as the ligand. The C415A mutant has also proven to be invaluable for other studies. Although unable to synthesize •NO, this mutant has a functional reductase domain. This has provided evidence for a novel regulatory mechanism of electron transfer mediated by calmodulin (see below). This mutant also aided us in the search for a novel metal binding site in the protein (see below). Other mutations have been carried out and several more are planned. We have recently succeeded in the expression of iNOS in E. coli (9). This isoform binds CaM essentially irreversibly, hence it is constitutively active. iNOS must be co-expressed with CaM as this isoform is highly unstable in the absence of CaM.

Function of the pterin cofactor - This remains a key unanswered question. Although it has been proposed that the function of the pterin involves dimerization of the enzyme, it has become clear that the pterin is not an absolute requirement in the dimerization process.  Experiments with pterin-free iNOS support a role for the pterin in the conversion of L-arginine to NG-hydroxy-L-arginine as well as a function in the conversion of NG-hydroxy-L-arginine to L-citrulline and •NO (9, 10).  Recent results obtained in collaboration with Dale Edmonson and Vincent Huynh at Emory University have shown that a pterin radical is formed during turnover by a single electron reduction of the ferrous-oxy complex (11).  Further characterization is underway.

Mechanistic studies - Experiments utilizing the so-called 'peroxide shunt' have provided insight into the conversion of NG-hydroxy-L-arginine to L-citrulline and •NO; additionally, these results suggest that the heme is not involved in the first step in the chemistry (12, 13). Current studies involving the chemistry catalyzed by individually expressed domains of the protein as well as studies using substrate analogues should have significant impact on our current understanding of the chemical steps in catalysis (14).

Function of Calmodulin (CaM) - It had been reported that CaM controls electron transfer from the reductase domain flavins to the heme, and increases the rate of flavin reduction (15). This is a novel regulatory role for this ubiquitous protein and one which we are actively pursuing. Our initial approach was to gain insight into the role of CaM in regulating the activity of iNOS (16). We have also been exploring the molecular details of this unusual method of regulation using Ca2+ binding site mutants of CaM. Through a collaboration with Dr. Kathy Beckingham at Rice University, we have obtained CaM mutants [these CaMs have specific point mutations (E to Q) in each of the four Ca2+ binding sites], and have used them to examine their effects on nNOS activity (17). Additionally, in collaboration with Drs. Henry Mosberg and Erik Zuiderweg at the University of Michigan, we are exploring structural aspects of the tight binding of CaM to iNOS by solution NMR of small iNOS-derived  peptides. 


References:

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  2. Nathan, C. (1992) FASEB J. 6, 3051-64.

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  5. Ludwig, M. L. and Marletta, M. A. (1999). A new decoration for nitric oxide synthase - a Zn(Cys)4 site. Structure 7: R73-R79.

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  6. Richards, M. K., and Marletta, M. A. (1994) Biochemistry 33, 14723-14732.

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  7. Perry, J. M., Moon, N., Zhao, Y., Dunham, W. R., and Marletta, M. A. (1998) Chemistry & Biology 5, 355-364.

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  8. Richards, M. K., Clague, M. J., and Marletta, M. A. (1996) Biochemistry 35, 7772-7780.

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  9. Rusche, K. M., Spiering, M. M. and Marletta, M. A. (1998). Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase. Biochemistry 37: 15503-15512.

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  10. Rusche, K. M. and Marletta, M. A. (2001). Reconstitution of pterin-free inducible nitric-oxide synthase. J. Biol. Chem. 276: 421-7.

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  11. Hurshman, A. R., Krebs, C., Edmondson, D. E., Huynh, B. H. and Marletta, M. A. (1999) Formation of a Pterin Radical in the Reaction of the Heme Domain of Inducible Nitric Oxide Synthase with Oxygen. Biochemistry 38: 15689-15696.

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  12. Pufahl, R. A., Wishnok, J. S., and Marletta, M. A. (1995) Biochemistry 34, 1930-1941.

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  13. Clague, M. J., Wishnok, J. S., and Marletta, M. A. (1997) Biochemistry 36, 14465-14473.

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  14. Marletta, M. A., Hurshman, A. R. and Rusche, K. M. (1998). Catalysis by nitric oxide synthase. Curr. Opin. Chem. Biol. 2: 656-663.

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  15. Abu-Soud, H. M., Feldman, P. L., Clark, P., and Stuehr, D. J. (1994) J. Biol. Chem. 269, 32318-32326.

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  16. Stevens-Truss, R., and Marletta, M. A. (1995) Biochemistry 34, 15638-15645.

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  17. Stevens-Truss, R., Beckingham, K., and Marletta, M. A. (1997) Biochemistry 36, 12337-12345.

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