JBC Oz Biosciences

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
QUICK SEARCH:   [advanced]


     

A more recent version of this article appeared on August 20, 2004
This Article
Right arrow Full Text (Accepted Manuscript)
Right arrow All Versions of this Article:
279/34/35412    most recent
M400872200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Konas, D. W.
Right arrow Articles by Stuehr, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Konas, D. W.
Right arrow Articles by Stuehr, D. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Papers In Press, published online ahead of print June 4, 2004
J. Biol. Chem, 10.1074/jbc.M400872200
Submitted on January 26, 2004
Revised on June 4, 2004
Accepted on June 4, 2004

The FAD-shielding residue Phe1395 regulates neuronal nitric oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain

David W. Konas, Keng Zhu, Manisha Sharma, Kulwant S. Aulak, Gary W. Brudvig, and Dennis J. Stuehr

Department of Immunology, Cleveland Clinic, Cleveland, OH 44195

Corresponding Author: stuehrd{at}ccf.org

Phe1395 stacks parallel to the FAD isoalloxazine ring in neuronal nitric oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe1395 was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP+ binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP+ with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP+ dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP+-bound state as compared to wild-type. We conclude that Phe1395 regulates nNOSr catalysis in two ways: It facilitates NADP+ release to prevent this step from being rate limiting and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN sub-domain that controls reactivity of its FMN cofactor in electron transfer.


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
R. P. Ilagan, M. Tiso, D. W. Konas, C. Hemann, D. Durra, R. Hille, and D. J. Stuehr
Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins
J. Biol. Chem., July 11, 2008; 283(28): 19603 - 19615.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C.-C. Wei, Z.-Q. Wang, J. Tejero, Y.-P. Yang, C. Hemann, R. Hille, and D. J. Stuehr
Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase
J. Biol. Chem., April 25, 2008; 283(17): 11734 - 11742.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. M. Haque, K. Panda, J. Tejero, K. S. Aulak, M. A. Fadlalla, A. T. Mustovich, and D. J. Stuehr
A connecting hinge represses the activity of endothelial nitric oxide synthase
PNAS, May 29, 2007; 104(22): 9254 - 9259.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z.-Q. Wang, R. J. Lawson, M. R. Buddha, C.-C. Wei, B. R. Crane, A. W. Munro, and D. J. Stuehr
Bacterial Flavodoxins Support Nitric Oxide Production by Bacillus subtilis Nitric-oxide Synthase
J. Biol. Chem., January 26, 2007; 282(4): 2196 - 2202.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Panda, M. M. Haque, E. D. Garcin-Hosfield, D. Durra, E. D. Getzoff, and D. J. Stuehr
Surface Charge Interactions of the FMN Module Govern Catalysis by Nitric-oxide Synthase
J. Biol. Chem., December 1, 2006; 281(48): 36819 - 36827.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Tiso, D. W. Konas, K. Panda, E. D. Garcin, M. Sharma, E. D. Getzoff, and D. J. Stuehr
C-terminal Tail Residue Arg1400 Enables NADPH to Regulate Electron Transfer in Neuronal Nitric-oxide Synthase
J. Biol. Chem., November 25, 2005; 280(47): 39208 - 39219.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Neeli, O. Roitel, N. S. Scrutton, and A. W. Munro
Switching Pyridine Nucleotide Specificity in P450 BM3: MECHANISTIC ANALYSIS OF THE W1046H AND W1046A ENZYMES
J. Biol. Chem., May 6, 2005; 280(18): 17634 - 17644.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.