Coupling of the Distal Hydrogen Bond Network to the Exogenous Ligand in Substrate-Bound, Resting State Human Heme Oxygenase

Abstract

Mammalian heme oxygenase, HO, possesses catalytically implicated distal ordered water molecules within an extended H-bond network, with one of the ordered water molecules (#1) providing a bridge between the iron-coordinated ligand and the catalytically critical Asp140, that, in turn, serves as an acceptor for the Tyr58 OH H-bond. The degree of H-bonding by the ligated water molecule and the coupling of this water molecule to the H-bond network are of current interest and are herein investigated by 1 H NMR. 2D NMR allowed sufficient assignments to provide both the H-bond strength and hyperfine shifts, the latter of which were used to quantify the magnetic anisotropy in both the ferric high-spin aquo and low-spin hydroxo complexes. The anisotropy in the aquo complex indicates that the H-bond donation to water #1 is marginally stronger than in a bacterial HO, while the anisotropy for the hydroxo complex reveals a conventional (d xz , d yz ) 1 ground state indicative of only moderate to weak H-bond acceptance by the ligated hydroxide. Mapping out the changes of the H-bond strengths in the network during the ligated water → hydroxide conversion by correcting for the effects of magnetic anisotropy, reveals a very substantial change in H-bond strength for Tyr58 OH, and lesser effects on nearby H-bonds. The effect of pH on the H-bonding network in human HO is much larger and transmitted much further from the iron than in a pathogenic bacterial HO. The implications for the HO mechanism of the H-bond of Tyr58 to Asp140 are discussed. KeywordsMagnetic anisotropy; hyperfine shifts; ligand deprotonation; heme oxygenase; H-bonding Heme oxygenase, HO, 1 is a widely distributed enzyme (1) that uses heme as a substrate and cofactor to cleave the heme via three intermediates, (2-6) as depicted in Figure 1. HOs occur in at least two forms in humans, a constitutive isozyme, designated hHO-2, and an inducible hHO-1. Both are membrane-bound, although the majority of the structure and function ‡ This research was supported by grants from the National Institutes of Health, GM62830 (GNL) and DK30297 (PROM).* lamar@chem.ucdavis.edu; phone: (530) 752-0958; FAX: (530) 752-8995.Supporting Information: Seven Figures (low-field 1 H NMR spectra, aromatic, aliphatic TOCSY, and inter-aliphatic NOESY for hHO-DMDH-H 2 O, and aromatic TOCSY, aromatic NOESY spectra and magnetic axes for hHO-DMDH-OH) and one table of chemical shifts. Total pages, 15. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations used: DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; PH, protohemin; DMDH, 2-,4-dimethyldeuterohemin; HO, heme oxygenase, hHO, human heme oxygenase#1; D140A-hHO, Asp140→Ala human heme oxygenase#1; NmHO, Neisseria meningitidis heme oxygenase; CdHO, Corynebacterium diphtheriae heme oxygenase; PaHO, Pseudomonas aeruginosa heme oxygenase; NOESY, two-dimensional nuclear Overhauser spectroscopy; TOCSY, two-dimensional total correlation spectroscopy; NIH Public Access Author ManuscriptBiochemistry. Author ma...

Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI

“…In contrast, the regiospecificity of classic HOs is achieved by steric restriction of the hydroperoxy ligand that forms during heme oxidation so that only the α-meso carbon is accessible for hydroxylation (6,8,9). In addition, the distal heme pockets of classic HOs also possess a group of ordered water molecules that direct protons to the catalytic center to stabilize the reactive hydroperoxy intermediate (8,(10)(11)(12)(13)(14). This water network is proposed to be required for catalysis by all classic HOs (11,(15)(16)(17)(18), but it is absent from the active sites of IsdG and IsdI (5,19).…”

Nuclear Magnetic Resonance as a Tool to Characterize the Interactome of Heme Proteins

“…NMR, in combination with other spectroscopies and molecular dynamics calculations, has been extensively used to learn about the heme degradation mechanism, both via the characterization of the heme electronic structure and degradation products with extensive use of 13 C-NMR on selectively enriched hemes and via the definition of conformational and dynamical properties of the polypeptide part with the classical bio-NMR approaches. [98][99][100][101][102][103][104][105] Other proteins that convey heme across the bacterial cells are the ironregulated surface determinant proteins (IsD) from gram-positive bacteria. 106 IsdC in Staphylococcus aureus is the protein through which the heme passes across the cell wall; its NMR structure in the apo form and in the complex with the zincsubstituted protoporphyrin IX has been solved.…”

Solution 1H NMR characterization of substrate-free C. diphtheriae heme oxygenase: Pertinence for determining magnetic axes in paramagnetic substrate complexes

“…[18-20] In mammalian HOs [11, 12] and HmuO, [8, 9] a key homologous Asp (140 in mammalian HO, 136 in HmuO) is at the center of the coupling between the exogenous ligand and the distal H-bond network via the ordered water molecules. In spite of significant sequence [5] and structural [8, 9, 11] homology, particularly in the H-bond network, [18, 19] the Asp mutation completely abolished activity in mammalian HO, [21, 22] but only reduced it to one-half in HmuO.…”

“…[24] A simple system for examining the coupling of the exogenous ligand to the H-bond network through the ordered water molecules is the equilibrium between the resting-state, high-spin, aquo, and the low-spin, hydroxo complexes, where simple deprotonation of the ligated water within a conserved molecular structure converts the H-bond donor ligated water molecule into an H-bond acceptor hydroxide ligand. A recent 1 H 2D NMR study [20] has shown that, upon deprotonation of the axial water in human HO, hHO-1, the low-field bias of the Tyr58 OH, which is a strong H-bond donor to the catalytically critical Asp140, moves ~2.7 ppm to lower field, suggesting a very substantial increase in the Tyr58 H-bond donor strength in the hydroxide complex. However, the chemical shift changes cannot be directly related to changes in H-bonding, since both complexes are paramagnetic and protons can experience significant dipolar shifts, [25, 26] δ dip , within ~12Å of the iron.…”

“…However, the chemical shift changes cannot be directly related to changes in H-bonding, since both complexes are paramagnetic and protons can experience significant dipolar shifts, [25, 26] δ dip , within ~12Å of the iron. The H-bond strength is reflected only in the diamagnetic contribution, δ DSS (dia*), to the observed chemical shift for a labile proton in a complex, δ DSS (obs), given by: [18-20] $${\mathrm{normal\delta }}_{\mathrm{DSS}}(\mathrm{dia}\ast )={\mathrm{normal\delta }}_{\mathrm{DSS}}(\mathrm{obs})­{\mathrm{normal\delta }}_{\mathrm{dip}}$$…”