Direct measurement by laser flash photolysis of intramolecular electron transfer in a two-domain construct of murine inducible nitric oxide synthase.
Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.
Feng
Changjian
C
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
Thomas
Clayton
C
Holliday
Michael A
MA
Tollin
Gordon
G
Salerno
John C
JC
Ghosh
Dipak K
DK
Enemark
John H
JH
D000818:Animals; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010782:Photolysis; D017434:Protein Structure, Tertiary
D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D009249:NADP; D005486:Flavin Mononucleotide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse