H-NOX (Heme Nitric oxide and/or OXygen) binding proteins are found in organisms from bacteria to humans and include soluble guanylate cyclase (sGC), the principle mammalian receptor for nitric oxide (NO). The ferrous heme of sGC selectively binds NO - completely excluding oxygen (O2) binding - and resists oxidation in air. These features allow sGC to respond to NO even in aerobic cellular conditions. In the present study, we investigated the ability of O2 to access the sGC heme pocket by incorporating a phosphorescent ruthenium porphyrin into two sGC domain truncations to serve as an internal O2 sensor. A crystal structure obtained for a homologous H-NOX domain suggests that incorporation of the ruthenium porphyrin does not alter the H-NOX fold. In the sGC domains, differences were observed in both emission intensities and lifetimes +/- O2, providing the first direct evidence that O2 accesses the sGC heme pocket. In addition, the extent of O2 quenching was found to parallel oxidation rates in the domains. Together these findings highlight the complexities of O2 discrimination in sGC and point to the role of higher protein structure in partially protecting the heme from O2.View Large Image | View Hi-Res Image | Download PowerPoint Slide