Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential

Huiying Li,Aditi Das,Hiruy Sibhatu,Joumana Jamal,Stephen G Sligar,Thomas L Poulos

doi:10.1074/jbc.m806949200

Abstract

In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give Delta G810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. Delta G810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of Delta G810 is insensitive to calmodulin binding. In addition, calmodulin binding to Delta G810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in Delta G810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity.

Highlights

Whether a red anionic or a blue neutral semiquinone can be stabilized in a particular flavoprotein is controlled by the protein-FMN interactions around the FMN N5 position
In the O-up conformation a peptide carbonyl oxygen from the loop accepts an H-bond from N5 of FMN. This interaction shifts the pKa of N5 to Ͼ13.0 [22] stabilizing the N5-protonated, blue neutral form of the FMN semiquinone in flavodoxins. This is why the semiquinone is more stable than the hydroquinone and why the ox/sq couple exhibits a higher redox potential than the sq/hq couple
The FMN binding loop in both cytochrome P450 reductase (CPR) and nitric-oxide synthase (NOS) is in the O-up conformation similar to what was described for the FMN semiquinone in flavodoxin structures

Summary

EXPERIMENTAL PROCEDURES

REACTION 1 sient, red anionic form rather than the air-stable, blue neutral radical seen in mammalian CPR. The wild-type FMN domain protein bound to free extract was obtained by ultracentrifugation at 100,000 ϫ g the nickel column often showed a blue color due to its air-stable for 1 h and loaded onto a small phenyl-Sepharose column semiquinone, whereas the ⌬G810 mutant FMN domain exhib- (2.6 ϫ 4.0 cm, GE Healthcare) pre-equilibrated with the same ited a bright orange color. The Q column was connected to an AKTA system (GE Health- The cuvette was sealed with an air tight septum through which care), being further washed with 5 bed volumes of phosphate the Ag/AgCl reference electrode, the gold working electrode, buffer containing 100 mM NaCl before the protein elution with and gas-tight Hamilton syringe were inserted. For the FMN domain of the wild-type nNOS, the absorbance changes at wavelengths 456 nm and 590 nm were plotted against the measured potential of the gold electrode. All these five variables are determined by least-squares fitting

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DISCUSSION