Stabilization and characterization of a heme-oxy reaction intermediate in inducible nitric-oxide synthase

Abstract

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that N-hydroxylate L-arginine (L-Arg) to make NO. NOS contain a unique Trp residue whose side chain stacks with the heme and hydrogen bonds with the heme thiolate. To understand its importance we substituted His for Trp188 in the inducible NOS oxygenase domain (iNOSoxy) and characterized enzyme spectral, thermodynamic, structural, kinetic, and catalytic properties. The W188H mutation had relatively small effects on l-Arg binding and on enzyme heme-CO and heme-NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild-type iNOSoxy, indicating it decreased heme-thiolate electronegativity. The protein crystal structure showed that the His188 imidazole still stacked with the heme and was positioned to hydrogen bond with the heme thiolate. Analysis of a single turnover L-Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Its build up coincided kinetically with the disappearance of the enzyme heme-dioxy species and with the formation of a tetrahydrobiopterin (H4B) radical in the enzyme, whereas its subsequent disappearance coincided with the rate of l-Arg hydroxylation and formation of ferric enzyme. We conclude: (i) W188H iNOSoxy stabilizes a heme-oxy species that forms upon reduction of the heme-dioxy species by H4B. (ii) The W188H mutation hinders either the processing or reactivity of the heme-oxy species and makes these steps become rate-limiting for l-Arg hydroxylation. Thus, the conserved Trp residue in NOS may facilitate formation and/or reactivity of the ultimate hydroxylating species by tuning heme-thiolate electronegativity.

FIGURE 1.

Reaction scheme for the single turnover l-Arg hydroxylation of NOS enzymes. After formation of the ferrous dioxygen complex (I) the subsequent steps are fast and none of the three putative intermediates (II, III, and IV; dashed boxes) have been spectroscopically observed in single turnover reactions. See text for details.

FIGURE 2.

Spectral properties of the W188H iNOSoxy mutant in the presence of H₄B and l-Arg. Representative spectra for the enzyme in the oxidized and reduced states, as well as the Fe^II-CO, Fe^II-NO, and Fe^III-NO complexes are shown.

FIGURE 3.

Comparison of wild-type and W188H mutant iNOSoxy. Structures of wild-type (green, PDB 1NOD (10)) and W188H iNOSoxy (magenta) exhibit similar structures in the heme region. The orientation of the wild-type Trp¹⁸⁸ indole nitrogen and W188H ε nitrogen are consistent and both point toward the Cys¹⁹⁴ sulfide. Hydrogen bonds are shown between the Cys¹⁹⁴ sulfide and the Trp¹⁸⁸ indole nitrogen (magenta dashes), between the Cys¹⁹⁴ sulfide and the His¹⁸⁸ ε nitrogen (green dashes), and between Cys¹⁹⁴ sulfide and Gly¹⁹⁶ backbone nitrogen (yellow dashes) are marked. The peptidic nitrogen of Ile¹⁹⁵ is not in a proper geometry for a hydrogen bond with the sulfide but the partial positive charges of the N-H hydrogen may contribute electrostatic interactions with the thiolate. Throughout the figure nitrogen (blue) and oxygen (red) are colored separately for clarity. The H₄B molecule from the W188H structure is showed in cyan, the other H₄B molecule is omitted for clarity. The figure was generated using PyMOL (96).

FIGURE 4.

Determination of the redox potential of wild-type and W188H iNOSoxy proteins. The fraction of oxidized protein at each redox potential value was fitted to the Nernst equation. The fitted values are: iNOSwt, -261 ± 2 mV; W188H, -173 ± 2 mV.

FIGURE 5.

Stopped-flow analysis of the heme transitions during l-Arg hydroxylation by the W188H iNOSoxy mutant. Single turnover reactions were initiated by mixing anaerobic ferrous protein with air saturated buffer at 10 °C. A, spectra of the Fe^II, Fe^II-O₂, Intermediate, and Fe^III heme species as calculated from Specfit global analysis of the kinetic data. The wavelength of the Soret band peaks is indicated. B and C, concentration of the Fe^II, Fe^II-O₂, Intermediate, and Fe^III heme species versus time during the short (B) or longer times (C) as calculated from Specfit global analysis of the kinetic data.

FIGURE 6.

Formation of the H₄B radical in the W188H iNOSoxy single turnover reaction at 10 °C as monitored by EPR. Top, spectra at different time points. Bottom, fit of the radical concentration to B according to an A → B → C reaction scheme.

FIGURE 7.

Stoichiometry of NOHA formation by wild-type and W188H iNOSoxy in the single turnover reaction. Samples of wild-type or W188H iNOSoxy were prereduced with a stoichiometric amount of dithionite and then mixed with aerobic buffer and incubated for 10 min (see text for details). The amount of NOHA formed from different starting concentrations of NOS was quantified by HPLC.

FIGURE 8.

Kinetics of product formation as determined by rapid-quench experiments and HPLC analysis. Anaerobic ferrous W188H was mixed with oxygen saturated buffer at 10 °C. Samples were quenched at different times and the concentrations of l-Arg (closed squares), NOHA (open circles), and citrulline (closed triangles) were determined by HPLC. Lines indicate the fit to a single exponential equation.