Rapid Calmodulin-dependent Interdomain Electron Transfer in Neuronal Nitric-oxide Synthase Measured by Pulse Radiolysis

Kazuo Kobayashi,Seiichi Tagawa,Simon Daff,Ikuko Sagami,Toru Shimizu

doi:10.1074/jbc.m102537200

Abstract

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.

Highlights

Nitric oxide (NO)1 is a key signaling molecule in a diverse array of cellular processes
The nitric-oxide synthases (NOS) enzyme is composed of an oxygenase domain with a thiol-coordinated heme active site similar to that of cytochrome P450 (P450), and an electron transfer domain related to NADPH-cytochrome P450 reductase which binds FMN and FAD (8 –12)
The initial transient increase in absorbance indicated the formation of the methyl-3-carbamoyl pyridinium (MCP) radical with an absorption maximum at 420 nm

Summary

EXPERIMENTAL PROCEDURES

Materials—Reagents were purchased from Sigma or Wako Pure Chemicals (Osaka, Japan), and were of the highest guaranteed grade and without purification. The oxygenase domain of the wild-type enzyme (Wildox) was prepared as previously mentioned [32]. The concentration of nNOS was determined optically from the [CO-reduced] Ϫ [reduced] difference spectrum using ⌬⑀444–467 nm ϭ 55 mMϪ1 cmϪ1 This ⌬⑀ value was estimated by the pyridine hemochromogen method [33] assuming that one heme is bound to one subunit of the enzyme. In the case of Wildox, the equilibrium buffer contained 0.2 M KCl. Solutions of nNOS contained 2 mM MCP and 0.1 M tert-butyl alcohol (for scavenging OH radicals) in 10 mM phosphate buffer (pH 7.4) and were deoxygenated in sealed cells by repeated evacuation and flushing with argon. The concentration of MCP radical generated by pulse radiolysis was estimated from the absorbance at 420 nm, using a molar extinction coefficient of 3300 MϪ1 cmϪ1 [35]. Optical absorption spectra were measured with a Hitachi U-3000 or a Shimadzu UV-2500 spectrophotometer

RESULTS

Electron Transfer in NO Synthase

DISCUSSION

Wild type ϩ