Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme |
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Authors: | Changjian Feng Andrea L Dupont Nickolas J Nahm Donald E Spratt James T Hazzard J Brice Weinberg J Guy Guillemette Gordon Tollin Dipak K Ghosh |
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Institution: | (1) College of Pharmacy, University of New Mexico, Albuquerque, NM 87131, USA;(2) Department of Chemistry, University of Waterloo, Waterloo, ON, N2L 3G1, Canada;(3) Department of Medicine, Duke University, VA Medical Centers, Durham, NC 27705, USA;(4) Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA |
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Abstract: | Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase
(NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN–heme IET in
a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along
with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808–3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme,
using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain.
The IET rate constants for the human and murine iNOS holoenzymes are 34 ± 5 and 35 ± 3 s−1, respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of
FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding
iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded
electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component
in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET
rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting
that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Electron transfer Nitric oxide synthase Laser flash photolysis Heme Flavin |
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