The heme in soluble guanylate cyclases (sGC) as isolated is ferrous, high-spin, and 5-coordinate. [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (ODQ) has been used extensively as a specific inhibitor for sGC and as a diagnostic tool for identifying a role for sGC in signal transduction events. Addition of ODQ to ferrous sGC leads to a Soret shift from 431 to 392 nm and a decrease in nitric oxide (NO)-stimulated sGC activity. This Soret shift is consistent with oxidation of the ferrous heme to ferric heme. The results reported here further define the molecular mechanism of inhibition of sGC by ODQ. Addition of ODQ to the isolated sGC heme domain [β1(1-385)] gave the same spectral changes as when sGC was treated with ODQ. EPR and resonance Raman spectroscopy was used to show that the heme in ODQ-treated β1(1-385) is indeed ferric. Inhibition of the NO-stimulated sGC activity by ODQ is due to oxidation of the sGC heme and not to perturbation of the catalytic site, since the ODQ-treated sGC has the same basal activity as untreated sGC (68 ± 12 nmol min-1 mg-1). In addition, ODQ-oxidized sGC can be re-reduced by dithionite, and this re-reduced sGC has identical NO-stimulated activity as the original ferrous sGC. Oxidation of the sGC heme by ODQ is fast with a second-order rate constant of 8.5 x 103 M-1 s-1. ODQ can also oxidize hemoglobin, indicating that the reaction is not specific for the heme in sGC versus that in other hemoproteins.