Reaction of neuronal nitric-oxide synthase with 2,6-dichloroindolphenol and cytochrome c3+: influence of the electron acceptor and binding of Ca2+-activated calmodulin on the kinetic mechanism

Biochemistry. 2001 Apr 17;40(15):4722-37. doi: 10.1021/bi0023495.


Binding of Ca(2+)-activated calmodulin (Ca(2+)-CaM) to neuronal nitric-oxide synthase (nNOS) increases the rate of 2,6-dichloroindolphenol (DCIP) reduction 2-3-fold and that of cytochrome c(3+) 10-20-fold. Parallel initial velocity patterns indicated that both substrates were reduced via two-half reactions in a ping-pong mechanism. Product and dead-end inhibition data with DCIP were consistent with an iso ping-pong bi-bi mechanism; however, product and dead-end inhibition studies with cytochrome c(3+) were consistent with the (two-site) ping-pong mechanism previously described for the NADPH-cytochrome P450 reductase-catalyzed reduction of cytochrome c(3+) [Sem, D., and Kasper, C. (1994) Biochemistry 33, 12012--12021]. Dead-end inhibition by 2'-adenosine monophosphate (2'AMP) was competitive versus NADPH for both electron acceptors, although the value of the slope inhibition constant, K(is), was 25-30-fold greater with DCIP as the substrate than with cytochrome c(3+). The difference in the apparent affinity of 2'AMP is proposed to result from a rapidly equilibrating isomerization step that occurs in both mechanisms prior to the binding of NADPH. Thus, initial velocity, product, and dead-end inhibition data were consistent with a di-iso ping-pong bi-bi and an iso (two-site) ping-pong mechanism for the reduction of DCIP and cytochrome c(3+), respectively. The presence Ca(2+)-CaM did not alter the proposed kinetic mechanisms. The activated cofactor had a negligible effect on (k(cat)/K(m))(NADPH), while it increased (k(cat)/K(m))(DCIP) and (k(cat)/K(m))(cytc) 4.5- and 23-fold, respectively.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 2,6-Dichloroindophenol / chemistry*
  • Adenosine Monophosphate / chemistry
  • Animals
  • Binding, Competitive
  • Calcium / chemistry
  • Calmodulin / chemistry*
  • Calmodulin / metabolism
  • Catalysis
  • Cytochrome c Group / antagonists & inhibitors
  • Cytochrome c Group / chemistry*
  • Electron Transport
  • Enzyme Inhibitors / chemistry
  • Kinetics
  • Models, Chemical
  • NADP / chemistry
  • Nitric Oxide Synthase / antagonists & inhibitors
  • Nitric Oxide Synthase / chemistry*
  • Nitric Oxide Synthase Type I
  • Oxidation-Reduction
  • Protein Binding
  • Rats
  • Substrate Specificity


  • Calmodulin
  • Cytochrome c Group
  • Enzyme Inhibitors
  • Adenosine Monophosphate
  • NADP
  • cytochrome c(3)
  • 2,6-Dichloroindophenol
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type I
  • Nos1 protein, rat
  • Calcium