A constitutively activated mutant of human soluble guanylyl cyclase (sGC): implication for the mechanism of sGC activation

Abstract

Heterodimeric alphabeta soluble guanylyl cyclase (sGC) is a recognized receptor for nitric oxide (NO) and mediates many of its physiological functions. Although it has been clear that the heme moiety coordinated by His-105 of the beta subunit is crucial for mediating the activation of the enzyme by NO, it is not understood whether the heme moiety plays any role in the function of the enzyme in the absence of NO. Here we analyze the effects of biochemical and genetic removal of heme and its reconstitution on the activity of the enzyme. Detergent-induced loss of heme from the wild-type alphabeta enzyme resulted in several-fold activation of the enzyme. This activation was inhibited after hemin reconstitution. A heme-deficient mutant alphabetaCys-105 with Cys substituted for His-105 was constitutively active with specific activity approaching the activity of the wild-type enzyme activated by NO. However, reconstitution of mutant enzyme with heme and/or DTT treatment significantly inhibited the enzyme. Mutant enzyme reconstituted with ferrous heme was activated by NO and CO alone and showed additive effects between gaseous effectors and the allosteric activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine. We propose that the heme moiety through its coordination with His-105 of the beta subunit acts as an endogenous inhibitor of sGC. Disruption of the heme-coordinating bond induced by binding of NO releases the restrictions imposed by this bond and allows the formation of an optimally organized catalytic center in the heterodimer.

Fig. 1.

Removal and reconstitution of heme regulates the basal activity of wild-type sGC. (A) Representative spectra of 3 μM sGC in 50 mM TEA, pH 7.4/200 mM NaCl treated with 0.1–0.5% Tween 20. a.u., arbitrary units. (B) After each Tween 20 treatment, basal activity was determined. Enzyme (3 μM) in 50 mM TEA, pH 7.4/200 mM NaCl treated with 0.5% Tween 20 was reconstituted with 0.5–6 μM hemin. (C) Difference spectra of obtained enzyme. (D) Basal activity of hemin-reconstituted enzyme (filled bars) was compared with the activity of PPIX-reconstituted sGC (line, open triangles). Data are representative of three independent experiments, with similar results performed in triplicate shown. Values are shown as means ± SD.

Fig. 2.

Basal activity of the mutantαβ^Cys-105 enzyme is comparable to the activity of NO-treated sGC. Specific activity of the purified mutant (filled bars) and wild-type (open bars) enzymes in the absence (basal) or presence of 100 μM SNP was determined. Data are shown as means ± SD of three independent experiments performed in triplicate. (Left Inset) UV-Vis spectra of 3 μM purified wild-type and mutant sGC. AU, arbitrary units. (Right) Coomassie blue staining of 5 μg of the wild-type (W) and mutant (M) sGC preparation. The size (in kilodaltons) of the molecular mass markers (PM) is indicated.

Fig. 3.

Hemin-dependent inhibition of the mutant enzyme. (A) αβ^Cys-105 (4 μM) was supplied with 0.5–20 μM hemin, and specific activity at each hemin concentration was determined and normalized to basal 7.6 ± 0.3 μmol/min per mg activity of the mutant specified as 100%. (Inset) Difference spectra of reconstituted enzyme recorded in 50 mM TEA, pH 7.4/200 mM NaCl. AU, arbitrary units. (B) Specific activity of the purified mutant was determined in the reaction buffers containing 1 mM DTT, 2μM hemin, or both. Data are shown as means ± SD of four independent experiments performed in triplicate.

Fig. 4.

Heme reconstitution of αβ^Cys-105 enzyme. (A) αβ^Cys-105 (4 μM) enzyme was supplied with 10 μM hemin and, after 15 min at 24°C, passed through a Hi-Trap desalting column (Amersham Biosciences). The UV-Vis spectra of reconstituted enzyme (solid line) were recorded in 50 mM TEA, pH 7.4, with 200 mM NaCl/10% glycerol. The spectra of reconstituted αβ^Cys-105 oxidized with 5 μM K₃[Fe(CN)₆] (interrupted line) or reduced by several grains of dithionate (dotted line) are shown. (Inset) Changes in the Soret region of dithionate-reduced hemin-reconstituted αβ^Cys-105 mutant (DTN, solid line) after 15 min of treatment with 50μM 3-(2-hydroxyl-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (interrupted line) or after exposure to CO (dotted line). post GF, post gel filtration; AU, arbitrary units.

Fig. 5.

Reconstitution of ferrous heme restores sensitivity of the mutant enzyme to NO and CO after DTT treatment. (A) Mutant enzyme treated with 1 mM DTT, 2 μM hemin, or both was assayed in the presence of SNP or under anaerobic conditions with CO-saturated reaction buffer. (B) Specific activity of the mutant enzyme exposed to 2 μM PPIX or 2 μM BAY41-2272 (BAY) was determined in the absence or presence of 1 mM DTT. The data for A and B are shown as means ± SD of three independent experiments performed in triplicate. (C) Heme-reconstituted mutant assayed in the presence of 1 mM DTT with or without 2 μM BAY41-2272 and treated with 100 μM SNP or with CO-saturated buffer. Representative data (means ± SD) of four independent experiments performed in triplicate are shown.

Fig. 6.

Schematic representation of heme-dependent regulation of wild type (A) and αβ^Cys-105 mutant (B). Catalytic center is schematically represented as a GTP molecule interacting with three structural elements. Maximal catalytic activity is achieved when all three elements are firmly interacting with GTP: activated conformation. Insertion of the heme and formation of a coordinating bond with His-105 (wild type) or Cys-105 (mutant) induces changes in the regulatory domain, affects the optimal structure of catalytic center, and results in a restricted or inhibited conformation. Activated conformation can be achieved by binding of NO to the heme moiety and disruption of the heme-coordinating bond. Removal of heme in wild-type enzyme results in an intermediate attenuated conformation, which in theαβ^Cys-105 mutant mimics activated conformation due to a stabilizing effect of a DTT-sensitive modification or interaction designated as “X.” Binding of PPIX to a heme-deficient enzyme (wild type or mutant) also results in activated conformation.