Abstract
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 k(on) constants at 4.5°C: 6.6×10(8)M(-1)s(-1) for nNOS(726-749), 2.9×10(8)M(-1)s(-1) for eNOS(492-511) and 6.1×10(8)M(-1)s(-1) for iNOS(507-531), 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 Ca(2+) chelation with ETDA: 3.7s(-1) for nNOS(726-749), 4.5s(-1) for eNOS(492-511), and 0.063s(-1) for iNOS(507-531). 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 iNOS(507-531) was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.