A molecular basis for NO selectivity in soluble guanylate cyclase Elizabeth M Boon 1 , Shirley H Huang 2 & Michael A Marletta 1–3 Soluble guanylate cyclases (sGCs) function as heme sensors that selectively bind nitric oxide (NO), triggering reactions essential to animal physiology. Recent discoveries place sGCs in the H-NOX family (heme nitric oxide/oxygen-binding domain), which includes bacterial proteins from aerobic and anaerobic organisms. Some H-NOX proteins tightly bind oxygen (O 2 ), whereas others show no measurable affinity for O 2 , providing the basis for selective NO signaling in aerobic cells. Using a series of wild-type and mutant H-NOXs, we established a molecular basis for ligand discrimination. A distal pocket tyrosine is requisite for O 2 binding in the H-NOX family. These data suggest that sGC uses a kinetic selection against O 2 ; we propose that the O 2 dissociation rate in the absence of this tyrosine is fast and that a stable O 2 complex does not form. Soluble guanylate cyclase is a heme sensor protein that selectively binds NO at the heme iron, activating the enzyme to convert guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) 1 . cGMP subsequently mediates a number of important physiological processes, including smooth muscle relaxation and neurotransmission. NO, which is present in target cells at a concentration of B10 nM, is an effective signaling molecule even in the aerobic environment of the cell (intracellular O 2 concentrations are B20–40 mM) because sGC overcomes the inherent affinity of heme for O 2 , thereby preventing a stable iron-oxygen bond and avoiding this highly unfavorable compe- tition. Because the concentration of O 2 is much higher than that of NO in eukaryotic cells, if sGC bound O 2 even weakly, there would be competition for the heme between NO and O 2 , resulting in non- selective formation of cGMP, blocking of the specific NO signal, or both. This ability of sGC not to bind O 2 is even more notable in light of the fact that the heme in sGC is identical to that in the O 2 storage and transport globin proteins—that is, they use the same protopor- phyrin IX, oxidation state and proximal histidine ligand. The mole- cular factors that allow sGC to discriminate between the apolar diatomic gases, NO and O 2 , are of great interest considering that this discrimination is central to selective biological responses to NO. The marked selectivity against O 2 exhibited by sGC is highlighted by the discovery of a new family of heme proteins in prokaryotes with substantial homology (15–40% identity) to the heme domain from sGC (Fig. 1) 2–4 . Through cloning and initial spectroscopic characteri- zation of several of these sGC-like heme domains, our laboratory found that members of this family from facultative aerobes are spectroscopically similar to sGC, as predicted, forming 5-coordinate NO complexes and rigorously excluding O 2 as a ligand 3 . The predicted heme sensor domain from the obligate anaerobe Thermoanaerobacter tengcongensis, however, is spectroscopically similar to the globins, forming a stable oxygen complex and a 6-coordinate NO complex 3 . The family is named the H-NOX domain because spectroscopic results indicate that, using the same protein fold (based on sequence align- ment) and an identical heme cofactor, some members of the H-NOX family form a tight complex with O 2 while others, like sGC, can use NO as a ligand by selectively excluding O 2 . We recently reported the structure of the H-NOX domain from the anaerobe T. tengcongensis (Tt H-NOX), an O 2 -binding member of this family, solved to a resolution of 1.77 A ˚ (ref. 4). One of the prominent features of the O 2 -bound structure of the Tt H-NOX domain is the presence of a hydrogen-bonding network around the bound O 2 molecule (Fig. 1). Tyr140 is involved in a 2.74-A ˚ hydrogen bond to the bound O 2 , and Asn74 and Trp9 are involved in 2.89-A ˚ and 2.79-A ˚ hydrogen bonds to the phenolic oxygen of Tyr140, respectively. H-NOX sequence alignments show that all three of these residues are found only in anaerobic members of the H-NOX family, which, according to results with Tt H-NOX, are predicted to bind O 2 . These residues are absent in all of the H-NOX domains from facultative aerobes and eukaryotes (sGCs) that do not bind O 2 (Fig. 1). Thus, these residues became clear choices for mutagenesis studies aimed at determining the molecular factors involved in ligand discrimination within the H-NOX family. In this report, using the crystal structure as a guide, we examined a series of mutant proteins to develop a structure-function relationship for O 2 binding in the H-NOX family. RESULTS The role of a distal tyrosine Initially, we chose Tt H-NOX Y140L and W9F mutations, as well as the double mutant W9F Y140L, for investigation of their role in O 2 binding to Tt H-NOX. These mutants were generated by standard methods, expressed, purified and spectroscopically characterized as previously reported for wild-type Tt H-NOX 3 . The structurally conservative Y140F mutant was also generated. Tt Y140F is Published online 24 May 2005; doi:10.1038/nchembio704 1 Department of Chemistry, 2 Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA. 3 Division of Physical Biosciences, Lawrence Berkeley National Lab, Berkeley, California 94720, USA. Correspondence should be addressed to M.M. (marletta@berkeley.edu). NATURE CHEMICAL BIOLOGY VOLUME 1 NUMBER 1 JUNE 2005 53 ARTICLES © 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology