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Electron transfer in a human inducible nitric oxide synthase oxygenase/FMN construct co-expressed with the N-terminal globular domain of calmodulin |
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| Authors: | Changjian Feng Weihong Fan J. Guy Guillemette Gordon Tollin |
| Affiliation: | a College of Pharmacy, University of New Mexico, Albuquerque, NM 87131, USA b Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 c Department of Medicine, Duke University and VA Medical Centers, Durham, NC 27705, USA d Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA |
| Abstract: | The FMN-heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a truncated calmodulin (CaM) construct that includes only its N-terminal globular domain consisting of residues 1-75, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly fourfold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca2+ depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca2+-dependent reorganization of the truncated CaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of the IET. |
| Keywords: | NO, nitric oxide NOS, nitric oxide synthase iNOS, inducible NOS nNOS, neuronal NOS CaM, calmodulin NCaM, truncated N-terminal globular domain of calmodulin consisting of residues 1-75 oxyFMN, bi-domain NOS construct in which only the heme-containing oxygenase and FMN domains along with the CaM binding region are present iNOS oxyFMN-NCaM, iNOS oxyFMN construct co-expressed with NCaM IET, intraprotein electron transfer dRF, 5-deazariboflavin H4B, 6R-5,6,7,8-tetrahydrobiopterin |
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