UC Berkeley

Nitric oxide (NO) signaling in mammals occurs through activation of the soluble isoform of guanylate cyclase (sGC), which results in diverse physiological processes such as blood vessel dilation and neurotransmission. sGC is a heterodimeric heme protein that has evolved to be a specific sensor for NO. Although sGC contains the same histidyl-ligated porphyrin as the globins, it has no measureable affinity for oxygen and does not oxidize in air. These unusual features facilitate selective and robust activation by NO in aerobic cellular contexts. Questions regarding the mechanism of ligand discrimination in sGC led to the identification of sGC heme domain homologues in many different organisms where these proteins function as NO and/or oxygen sensors for gas-mediated signaling pathways. The members of the protein family have been subsequently termed Heme Nitric oxide/OXygen binding (H-NOX) domains due to their divergent ligand-binding properties. Functional characterization of H-NOX domains from prokaryotes has provided important clues about the structural features that control ligand discrimination across the H-NOX family. However, additional fundamental questions remain about the influence of protein structure on heme chemistry.

Extensive studies on the globins, as model heme proteins, have established the functional importance of heme protein topological features in modulating gas diffusion to and from the heme site. In globins, deep gas pockets around the porphyrin transiently capture and release oxygen, tuning oxygen affinity for transport, delivery, and storage in diverse physiological environments. Although mechanisms of gas binding in H-NOX proteins have been a subject of intense investigation, there is little knowledge regarding the functional role of protein structure in modulating gas diffusion. Using X-ray crystallography with xenon and kinetic measurements, a tunnel network that extends between the solvent and interior heme site was mapped in a prokaryotic H-NOX domain. Hindering gas diffusion through the tunnels has important consequences on diatomic gas affinity. This suggests that protein tunnels in H-NOX proteins may play functional roles in tuning gas-mediated signaling.

Unlike isolated H-NOX domains, mammalian sGC is a structurally more complex, multi-domain protein. The ability of the sGC heme to resist oxidation in air is unique among histidyl-ligated heme proteins and essential for maximal NO-induced activation. Speculations have been made about how the sGC heme resists oxidation, but no study has systemically addressed the structural and electronic factors that contribute to this critical property. To probe the accessibility of oxygen to the heme site, we sought to substitute the native heme of sGC with an unnatural porphyrin displaying emission that is quenched by oxygen. Using an expression-based methodology, a phosphorescent Ru porphyrin was incorporated in sGC constructs of varying lengths. Emission quenching results suggest that oxygen diffusion to the full-length sGC heme site is significantly hindered compared to smaller sGC constructs. Limited oxygen accessibility, combined with heme electronic factors, appear to serve as important evolutionary solutions in sGC to protect the heme cofactor under aerobic conditions.

Originally to probe sGC function, the strategies used to generate oxygen-sensing proteins have been applied to create a new family of H-NOX-based molecular sensors. Substitution of the native heme in bacterial H-NOX proteins with unnatural porphyrins has proven to be a promising strategy to develop stable protein agents for biological imaging applications. Heme proteins are highly tunable frameworks that coordinate porphyrins with high fidelity and specificity. These properties have been exploited to design heme protein-based sensors with tailored functionalities for enhancing porphyrin bioavailability. Initial work on making optical oxygen sensors has evolved into recent efforts to generate H-NOX-based MRI contrast agents with high relaxivities for future use in deep-tissue imaging. Thus, through substitution of the native heme group with unnatural porphyrins, new sensing properties have been built into the H-NOX family.