Abstract
Intraprotein interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme centers is an obligatory step in nitric oxide synthase (NOS) enzymes. An isoform-specific pivotal region near Leu406 in the heme domain of human inducible NOS (iNOS) was proposed to mediate the FMN-heme domain–domain alignment (J Inorg Biochem 153:186–196, 2015). The FMN-heme IET rate is a measure of the interdomain FMN/heme complex formation. In this work, the FMN-heme IET kinetics in the wild type (wt) human iNOS oxygenase/FMN (oxyFMN) construct were directly measured by laser flash photolysis with added synthetic peptide related to the pivotal region, in comparison with the wt construct alone. The IET rates were decreased by the iNOS HKL peptide in a dose-saturable fashion, and the inhibitory effect was abolished by a single L406 → E mutation in the peptide. A similar trend in change of the NO synthesis activity of wt iNOS holoenzyme by the peptides was observed. These data, along with the kinetics and modeling results for the L406T and L406F mutant oxyFMN proteins, indicated that the Leu406 residue modulates the FMN-heme IET through hydrophobic interactions. Moreover, the IET rates were analyzed for the wt iNOS oxyFMN protein in the presence of nNOS or eNOS-derived peptide related to the equivalent pivotal heme domain site. These results together indicate that the isoform-specific pivotal region at the heme domain specifically interacts with the conserved FMN domain surface, to facilitate proper interdomain docking for the FMN-heme IET in NOS.
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Abbreviations
- NO:
-
Nitric oxide
- NOS:
-
NO synthase
- iNOS:
-
Inducible NOS
- nNOS:
-
Neuronal NOS
- eNOS:
-
Endothelial NOS
- IET:
-
Intraprotein interdomain electron transfer
- CaM:
-
Calmodulin
- dRF:
-
5-Deazariboflavin
- H4B:
-
6R-5,6,7,8-Tetrahydrobiopterin
- l-Arg:
-
l-arginine
- oxyFMN:
-
A truncated bi-domain oxygenase/FMN construct of NOS that consists of only the heme-containing oxygenase domain and the FMN domain, along with the CaM-binding linker in between the two domains
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Acknowledgements
This work was supported by grants from the National Institutes of Health (GM081811 and GM133973 to C.F.).
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Supplementary file1 Primers for constructing the iNOS mutants; the FMN-heme IET and NO production rates of human iNOS proteins in the presence of the synthetic peptide. (PDF 111 kb)
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Zheng, H., Li, J. & Feng, C. An isoform-specific pivot modulates the electron transfer between the flavin mononucleotide and heme centers in inducible nitric oxide synthase. J Biol Inorg Chem 25, 1097–1105 (2020). https://doi.org/10.1007/s00775-020-01824-w
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DOI: https://doi.org/10.1007/s00775-020-01824-w