Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain

Abstract

Interior topological features, such as pockets and channels, have evolved in proteins to regulate biological functions by facilitating the diffusion of biomolecules. Decades of research using the globins as model heme proteins have clearly highlighted the importance of gas pockets around the heme in controlling the capture and release of O 2 . However, much less is known about how ligand migration contributes to the diverse functions of other heme protein scaffolds. Heme nitric oxide/oxygen binding (H-NOX) domains are a conserved family of gas-sensing heme proteins with a divergent fold that are critical to numerous signaling pathways. Utilizing X-ray crystallography with xenon, a tunnel network has been shown to serve as a molecular pathway for ligand diffusion. Structure-guided mutagenesis results show that the tunnels have unexpected effects on gas-sensing properties in H-NOX domains. The findings provide insights on how the flux of biomolecules through protein scaffolds modulates protein chemistry.

Structural insights into the role of iron–histidine bond cleavage in nitric oxide-induced activation of H-NOX gas sensor proteins

“…Notably, due to the coupling of the heme and protein conformations, it is then postulated that this proximal pocket hydrogen-bonding network aids in maintaining the protein in the basal state conformation. Furthermore, a similar mode of water coordination in the proximal pocket was also observed in Ns H-NOX (12,21) and has been hypothesized to be an important feature in H-NOX activation (22).…”

“…This water molecule has also previously been observed in Ns H-NOX and is postulated to be a general feature of H-NOX proteins from facultative anaerobes and sGC (12,21). Evidence has been provided for this water molecule serving a similar role in sGC, wherein mutation of the proposed water-coordinating residue in sGC, D102, to an alanine drastically affected heme binding and activation of the enzyme.…”

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

“…In hydrogenases, gas channels have been reported to selectively filter molecules such as oxygen and carbon monoxide to prevent inactivation at the active site (12,17,35). Recent investigation of heme nitric oxide/oxygen binding domains has suggested that tunnels not only direct gases to the heme iron but also modulate reversible gas binding (16). Importantly, our work is the first to directly implicate an oxygen channel in regulating regio-and stereospecificity by explicitly showing the production of alternate product isomers upon obstruction of the channel.…”

“…By contrast, native SLO-1 produces the 13S-hydroperoxide product (13S-HPOD) in greater than 90% yield for reaction of WT enzyme under optimal conditions. The precise mechanism by which SLO-1, as well as other lipoxygenases, maintain the regio-and stereospecificity of product peroxidation has engendered a variety of proposals that include: (i) an alteration in substrate binding (from head to tail first) as a means of reversing the enantiomeric specificity of O 2 addition to a fixed carbon center; (ii) the interchange of a single active site residue between Gly and Ala to alter the position (but not the face) of O 2 attack; and (iii) a directed movement of O 2 through the protein matrix toward a spatially defined position of substrate that is capable of simultaneously controlling both the position and face of O 2 attack on a substrate-derived free radical intermediate (4,16,18).Although all three of the above factors may play a role in the evolution of lipoxygenases that catalyze the formation of stereo-and regiochemically distinct hydroperoxides, there remains the generic question of how each enzyme manages a transit of O 2 from bulk solvent toward the reactive carbon of buried substrate. The x-ray structure of SLO-1 is quite informative in this regard, implicating a putative gas channel that appears "crimped" in its middle at the residue Ile 553 (18).…”

Discovery of stimulator binding to a conserved pocket in the heme domain of soluble guanylyl cyclase

“…While a highresolution structure of an sGC H-NOX domain has not been reported, crystal structures for several bacterial homologues are known, including those from Caldanaerobacter subterraneus (30), also known as Thermoanaerobacter tengcongensis (22,31), Nostoc sp. 7120 (32,33) and Shewanella oneidensis (34). These structures display the same overall fold and provide a solid scaffold for understanding H-NOX structure in sGC function (reviewed in (4,5,23)).…”