Neuronal Nitric-oxide Synthase Mutant (Ser-1412 → Asp) Demonstrates Surprising Connections between Heme Reduction, NO Complex Formation, and Catalysis

Subrata Adak,Jérôme Santolini,Svetlana Tikunova,Qian Wang,J David Johnson,Dennis J Stuehr

doi:10.1074/jbc.m006857200

Subrata Adak, Jérôme Santolini + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m006857200

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Abstract

Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.

Highlights

Rat neuronal Nitric oxide (NO) synthase contains an Akt-dependent phosphorylation motif in its reductase domain
The mutant did not synthesize NO without CaM and with CaM had a rate of NO synthesis that was 25% diminished compared with wild-type enzyme (Table I)
We conclude that the mutant displays slower NO synthesis, but this cannot be explained by defects in oxygenase domain catalysis or electron transfer through the reductase domain

Summary

EXPERIMENTAL PROCEDURES

Materials—All regents and materials were obtained from Sigma or sources previously reported (19). Reactions were initiated by rapid mixing an anaerobic, buffered, CO-saturated solution containing 50 ␮M NADPH with an anaerobic CO-saturated solution containing wild-type or mutant nNOS (2 ␮M), 40 mM EPPS buffer, pH 7.6, 10 ␮M H4B, 0.3 mM dithiothreitol, 5 mM Arg, 4 ␮M CaM, or 20 ␮M soybean CaM proteins, and 1 mM Ca2ϩ. To simulate percentage of ferrous-NO complex achieved at steady state, simulations were run using a 1-ms time step for each heme reduction rate obtained with CaM and the ScaM proteins. For O2 reaction with the Fe(II) heme-NO complex we used a pseudo-first order rate constant (0.19 sϪ1) that was reported for nNOS by Adak et al (7) at a single O2 concentration (half air-saturated) at 10 °C. We varied this rate constant in direct proportion to the simulated O2 concentration

RESULTS

DISCUSSION

45 Ϯ 3 69 Ϯ 5 35 Ϯ 3 28 Ϯ 2