Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes

Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742–8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast kon constants at 4.5 °C: 6.6 × 108 M− 1 s− 1 for nNOS726–749, 2.9 × 108 M− 1 s− 1 for eNOS492–511 and 6.1 × 108 M− 1 s− 1 for iNOS507–531, 3–4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca2+ chelation with ETDA: 3.7 s− 1 for nNOS726–749, 4.5 s− 1 for eNOS492–511, and 0.063 s− 1 for iNOS507–531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS507–531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.

Target peptides from all three NOS bind holo fluorescence labeled calmodulin with kon > 1 × 108 M− 1 s− 1 (labeling sites: red). Apo calmodulin dissociates from nNOS peptide or eNOS peptide at 3.7–4.5 s− 1 but binds to iNOS peptide, and conformation of the complex rearranges at 0.063 s− 1.Figure optionsDownload as PowerPoint slideHighlights
► The binding kinetics of Alexa 350 labeled calmodulin to nitric oxide synthase target peptides in solution was measured using fluorescence stopped-flow.
► The binding of calmodulin to nitric oxide synthase is fast and unlikely a rate-limiting step in the catalysis of nitric oxide synthase.
► Only inducible nitric oxide synthase is capable of binding to both apo and holo calmodulin, but in different modes.