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. 2009 Aug 14;284(33):21788-21796.
doi: 10.1074/jbc.M109.014282. Epub 2009 Jun 15.

The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: interactions at ferrous heme and cysteine thiols

Thomas W Miller  1 , Melisa M Cherney  2 , Andrea J Lee  2 , Nestor E Francoleon  1 , Patrick J Farmer  3 , S Bruce King  4 , Adrian J Hobbs  5 , Katrina M Miranda  6 , Judith N Burstyn  2 , Jon M Fukuto  7
Affiliations

The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: interactions at ferrous heme and cysteine thiols

Thomas W Miller et al. J Biol Chem. .

Abstract

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols.

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Figures

FIGURE 1.
FIGURE 1.
Schematic of Angeli's salt (A) and NCTFA (B) decomposition to yield HNO.
FIGURE 2.
FIGURE 2.
sGC is activated by HNO donors. sGC (3.2 μg final: 0.11 μm) activity was measured in the presence of NO (DEANO) or HNO (NCTFA, Angeli's salt) donors for 16 min at 37 °C. Data represents the average of triplicate measurements reported as fold-activation over basal activity (no donor).
FIGURE 3.
FIGURE 3.
Activation of sGC by HNO donors is not due to NO. A, effect of desferoxamine on sGC activation. The effect of excess NO (DEANO) or HNO (NCTFA, Angeli's salt) donors (100 μm) on sGC (3.6 μg final) activity was measured in the presence or absence of 10 μm of the metal chelator (desferrioxamine, DFX) for 15 min at 37 °C. Data represents the average of triplicate measurements reported as specific activity (nmol of cGMP mg−1 min−1). B, effect of DTT on sGC activation. We measured sGC (19 μg final) activation by 100 μm NO (DEANO) or HNO (NCTFA, Angeli's salt) donors in the presence or absence of 10 mm DTT for 15 min at 37 °C. Data represents the average of triplicate measurements reported as specific activity (nmol of cGMP mg−1 min−1).
FIGURE 4.
FIGURE 4.
HNO activation of sGC depends on the presence of ferrous heme. A, effect of heme removal on sGC activation. We measured the ability of NO (DEANO) or HNO (NCTFA, Angeli's salt) donors to activate sGC with and without heme. Untreated (heme bound) and treated (heme removed) sGC (19 μg final) were assayed for activity in the presence of 100 μm donors for 15 min at 37 °C. Data represents the average of triplicate measurements reported as fold-activation over basal activity (no donor). B, effect of heme redox state on sGC activation. We compared the ability of NO (DEANO) or HNO (NCTFA, Angeli's salt) donors to activate reduced and oxidized sGC. sGC was treated with ferricyanide to oxidize heme and re-reduced with DTT. Ferrous (reduced, untreated), ferric (oxidized, treated with ferricyanide), and ferric + DTT (re-reduced, treated with ferricyanide then DTT) sGC (3.5 μg final) were assayed for activity in the presence of 100 μm donors for 12 min at 37 °C. Data represents the average of triplicate measurements reported as fold-activation over basal activity (no donor). C, effect of ferric heme and HNO on sGC activation. Heme was removed from sGC and activity was measured in the presence of Angeli's salt (100 μm) alone or Angeli's salt (100 μm) and hemin chloride (0.1 μm). Data represents the average of triplicate measurements reported as specific activity (nmol of cGMP mg−1 min−1).
FIGURE 5.
FIGURE 5.
HNO inhibits activated sGC in a thiol dependent manner. A, effect of HNO (Angeli's salt) on sGC activity. We measured sGC (19 μg final) activation by an HNO donor (Angeli's salt) in the presence or absence of 10 mm DTT for 15 min at 37 °C. Data represents the average of triplicate measurements reported as specific activity (nmol of cGMP mg−1 min−1). B, effect of HNO donors on PPIX-activated sGC. Heme was removed from sGC and it was incubated in the absence (control) or presence of PPIX (100 nm) for 5 min at 37 °C. Activity was measured in the absence of PPIX, the presence of PPIX alone, or PPIX co-incubated with DEANO (100 μm), Angeli's salt (100 μm), or NCTFA (100 μm). Data represents the average of triplicate measurements reported as specific activity (nmol of cGMP mg−1 min−1).
FIGURE 6.
FIGURE 6.
Proposed mechanisms for the effects of HNO on sGC activity. Note, the possible and/or likely products of sGC thiol oxidation by excess HNO are shown. The exact nature of the products and the sites of these cysteine thiols are not known.
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