An isoform-specific pivot modulates the electron transfer between the flavin mononucleotide and heme centers in inducible nitric oxide synthase

Abstract

Intraprotein interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme centers is an obligatory step in nitric oxide synthase (NOS) enzymes. An isoform-specific pivotal region near Leu406 in the heme domain of human inducible NOS (iNOS) was proposed to mediate the FMN-heme domain-domain alignment (J Inorg Biochem 153:186-196, 2015). The FMN-heme IET rate is a measure of the interdomain FMN/heme complex formation. In this work, the FMN-heme IET kinetics in the wild type (wt) human iNOS oxygenase/FMN (oxyFMN) construct were directly measured by laser flash photolysis with added synthetic peptide related to the pivotal region, in comparison with the wt construct alone. The IET rates were decreased by the iNOS HKL peptide in a dose-saturable fashion, and the inhibitory effect was abolished by a single L406 → E mutation in the peptide. A similar trend in change of the NO synthesis activity of wt iNOS holoenzyme by the peptides was observed. These data, along with the kinetics and modeling results for the L406T and L406F mutant oxyFMN proteins, indicated that the Leu406 residue modulates the FMN-heme IET through hydrophobic interactions. Moreover, the IET rates were analyzed for the wt iNOS oxyFMN protein in the presence of nNOS or eNOS-derived peptide related to the equivalent pivotal heme domain site. These results together indicate that the isoform-specific pivotal region at the heme domain specifically interacts with the conserved FMN domain surface, to facilitate proper interdomain docking for the FMN-heme IET in NOS.

Keywords: Electron transfer; Kinetics; Laser flash photolysis; Nitric oxide synthase.

Figure 1.

(A) A docking model of human iNOS oxyFMN at the redox state of [Fe(III)][FMNH⁻], taken from molecular dynamics simulations [28]. The heme domain in the same subunit is in purple (and labeled Heme A), and the bound CaM is green. Note that the FMN domain docks to the heme domain (light blue) of the other subunit (labeled Heme B) to allow for the inter-subunit FMN – heme IET. The heme and FMN cofactors are displayed in ball and stick. (B) A close-up view of the pivotal residues displayed in stick. (C) Sequence alignment of the pivotal regions in the three NOS isoforms. Note that the pivotal residues HKL in human iNOS heme domain (highlighted in purple) differs among the NOS isoforms (RKT and RTT for nNOS and eNOS, respectively), while the pairing residues in the FMN domain (e.g., E661, L662, Q665, E666 in human iNOS) are conserved (colored in orange).

Figure 2.

Transient absorbance traces at 580 nm obtained for the [Fe(II)–CO][FMNH•] form of wild type human iNOS oxyFMN in the presence of LETHKLASLW peptide. The IET process was monitored by the loss of absorbance of FMNH• at 580 nm, which decayed below the pre-flash baseline upon a laser excitation. Single exponential model was used to fit to the data, as shown in red solid line. Plot of residual from the fitting to a single exponential function is shown in the bottom panel; note that the fitting gives an excellent normal distribution of the residuals. Anaerobic solution contained 15 μM iNOS oxyFMN, 90 μM LETHKLASLW peptide, ~ 20 μM 5-deazariboflavin and 5 mM fresh semicarbazide in a pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 2 mM L-Arg, 20 μM H₄B, 1 mM Ca²⁺ and 10 % glycerol). The experiment was conducted at 21 °C.

Figure 3.

Plot of the observed FMN – heme IET rates of wild type human iNOS oxyFMN protein with added iNOS-HKL peptide (LETHKLASLW), iNOS-HKE peptide (LETHKEASLW), nNOS-RKT peptide (LETRKTASLW) or eNOS-RTT peptide (LETRTTASLW). At [peptide]:[iNOS] ratio of 6, inhibitory effect of the iNOS-HKL peptide on the kinetics has been saturated. Since the goal of these experiments is to compare the inhibitory effects/trend of the various peptides, it is not necessary to examine all peptides at other lower concentrations.

Figure 4.

Plot of the NO production rates of wild type human iNOS holoprotein in the presence of various concentrations of the iNOS-HKL peptide (LETHKLASLW), iNOS-HKE peptide (LETHKEASLW), nNOS-RKT peptide (LETRKTASLW) or eNOS-RTT peptide (LETRTTASLW).

Figure 5.

Comparison of the interactions between the heme domain 406 residue and the FMN domain L662 residue (colored in magenta and yellow, respectively). The Leu406 residue was mutated to Thr, Phe, or Glu, using Chimera software.

Figure 6.

A close-up view of multiple domain-domain interacting sites in the docking complex model. The FMN/heme/CaM complex is shown in the same orientation as Figure 1. Only surface of the intra-subunit pivotal region (colored in parent color) is depicted, while the rest is displayed in line ribbon. Selected interacting residues are labelled for the CaM-heme and inter-subunit FMN-heme sites; these NOS and CaM interface residues have been reported in the literature [, –41].