Rapid calmodulin-dependent interdomain electron transfer in neuronal nitric-oxide synthase measured by pulse radiolysis.
Electron transfer within rat neuronal nitric-oxide synthase (nNOS) was investigated by pulse radiolysis. Radiolytically generated 1-methyl-3-carbamoyl pyridinium (MCP) radical was found to react predominantly with the heme of the enzyme with a second-order rate constant for heme reduction of 3 x 10(8) m(-1) s(-1). In the calmodulin (CaM)-bound enzyme a subsequent first-order phase was observed which had a rate constant of 1.2 x 10(3) s(-1). In the absence of CaM, this phase was absent. Kinetic difference spectra for nNOS reduction indicated that the second phase consisted of heme reoxidation accompanied by formation of a neutral flavin semiquinone, suggesting that it is heme to flavin electron transfer. Experiments with the heme proximal surface mutant, K423E, had no second phase, confirming that the mutation blocks interdomain electron transfer. With the autoinhibitory loop deletion mutant, Delta40, the slow phase was observed even in the absence of CaM consistent with the role of the loop in impeding interdomain electron transfer. The rate of heme to FMN electron transfer observed in the wild-type enzyme is approximately 1000 times faster than the FMN to heme electron transfer rate predicted during catalysis from kinetic modeling, suggesting that the catalytic process is slowed by kinetic gating.
Kobayashi
K
K
Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan. kobayasi@sanken.osaka-u.ac.jp
Tagawa
S
S
Daff
S
S
Sagami
I
I
Shimizu
T
T
D002147:Calmodulin / Q000378:metabolism*; D004579:Electron Transport; D005609:Free Radicals / Q000378:metabolism; D009154:Mutation; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010446:Peptide Fragments; D017434:Protein Structure, Tertiary; D011675:Pulse Radiolysis
D002147:Calmodulin; D005609:Free Radicals; D010446:Peptide Fragments; D009536:Niacinamide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C024058:N(1)-methylnicotinamide