Role of a Conserved Tyrosine Residue in the FMN-Heme Interdomain Electron Transfer in Inducible Nitric Oxide Synthase

Abstract

The interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme domains is essential in the biosynthesis of nitric oxide (NO) by the NO synthase (NOS) enzymes. A conserved tyrosine residue in the FMN domain (Y631 in human inducible NOS) was proposed to be a key part of the electron transfer pathway in the FMN/heme docked complex model. In the present study, the FMN-heme IET kinetics in the Y631F mutant and wild type of a bidomain oxygenase/FMN construct of human inducible NOS were determined by laser flash photolysis. The rate constant of the Y631F mutant is significantly decreased by ∼75% (compared to the wild type), showing that the tyrosine residue indeed facilitates the FMN-heme IET through the protein medium. The IET rate constant of the wild type protein decreases from 345 to 242 s^-1 on going from H₂O to 95% D₂O, giving a solvent kinetic isotope effect of 1.4. In contrast, no deuterium isotope effect was observed for the Tyr-to-Phe mutant. Moreover, an appreciable change in the wild type iNOS IET rate constant value was observed upon changing pH. These results indicate that the FMN-heme IET is proton coupled, in which the conserved tyrosine residue may play an important role.

Figure 1

Potential electron transfer pathway in a docked FMN/heme complex of human iNOS. The conserved Tyr631 is proposed to facilitate the FMN–heme IET through the protein medium: IET presumably takes place through FMN → Tyr631 → Trp372 → heme. An equivalent Trp366 in murine iNOS has been shown to participate in the IET. The FMN and heme domains are shown in blue and gray line ribbons, respectively. The docking surface residues on the FMN domain (E546 and E603) are colored in green, while the other residues and ligands are colored in element. The FMN and heme cofactors are shown in ball and stick.

Figure 2

Transient traces at 580 nm obtained for the [Fe(II)-CO][FMNH^•] form of Y631F human iNOS oxyFMN mutant flashed by 446 nm laser. The graph is a combined plot of two traces at 0 – 0.08 s and 0 – 1.8 s using a logarithmic timescale. Solid lines correspond to the best single-exponential fit to the data: upon a laser excitation, the absorption at 580 nm decays below the pre-flash baseline (red solid line, 0 – 0.08 s), which is due to the FMN–heme IET resulting in FMNH^• depletion, followed by a much slower recovery toward the baseline (green solid line, 0.08 – 1.8 s), which is due to the CO rebinding to Fe(II). The 0 – 0.08 s trace is an average of seven traces, while the 0 – 1.8 s trace is of a single trace. The sample temperature was set at 21 °C. Anaerobic solutions contained 10 μM Y631F iNOS oxyFMN, ~ 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).

Figure 3

Transient trace (0 – 0.018 s) at 580 nm obtained for the [Fe(II)-CO][FMNH^•] form of Y631F human iNOS oxyFMN protein flashed by 446 nm laser, in comparison with the trace of the wild type protein. Note that the wild type trace has become leveled within this time scale, while the Y631F mutant trace has not finished yet. The experimental conditions are the same as those in Figure 2.

Figure 4

Proton inventory plot of wild type human iNOS oxyFMN. The ratio of the IET rate constant at the indicated mole fraction of D₂O (n) to that in 100 % H₂O (k_n/k₀) is plotted versus n. R = 0.92 for the linear fit of the data using eq 4.

Figure 5

Transient trace at 465 nm obtained for the [Fe(II)-CO][FMNH^•] form of Y631F human iNOS oxyFMN protein in 95 % D₂O buffered solution flashed by 446 nm laser. Solid line corresponds to the best single-exponential fit to the data. The sample temperature was set at 21 °C. The experimental conditions are the same as those in Figure 2.

Figure 6

A possible PCET mechanism in which human iNOS Tyr631 acts as an intermediary proton shuttle between anionic FMN hydroquinone (FMNH⁻) and water solvent. The radical and negative charge symbols are colored in blue.