Complete Heme Proton Hyperfine Resonance Assignments of the Glycera dibranchiata Component IV Metcyano Monomer Hemoglobin

Abstract

Monomer hemoglobin component IV is one of three major myoglobin-like proteins found in the erythrocytes of the marine annelid Glycera dibranchiata. Unlike myoglobin, all three of these monomer hemoglobin components lack the distal histidine, which is replaced by leucine. This substitution alters the protein's functional properties due to its proximity to the heme ligand binding site. As the initial step toward a full NMR characterization of this protein, a complete set of self-consistent proton NMR assignments for the heme and the proximal histidine of the paramagnetic, metcyano form of native component IV (metGMH4CN) is presented. These assignments relied upon a combination of one- and two-dimensional NMR spectroscopy, including nonselective spin-lattice relaxation time measurements. The metcyano form has been chosen for several reasons: (1) The heme paramagnetism acts as an intrinsic shift reagent which aids in making individual resonance assignments for the heme and neighboring amino acids in the protein's ligand binding site. (2) Heme paramagnetism also enhances proton nuclear relaxation rates, thereby allowing two-dimensional NMR experiments to be carried out at very rapid repetition rates (i.e., 5 s-1). (3) The heme proton hyperfine resonance pattern for this paramagnetic form of wild-type monomer hemoglobin component IV provides an analytical reference for the integrity of the heme active site. This is anticipated to facilitate rapid analysis of subsequently produced recombinant derivatives of this protein. (4) The cyanide-ligated protein has a heme pocket structure similar to those of the O2- and CO-ligated forms of the physiologically important, reduced form of the protein, so that the heme and proximal histidine proton assignments will serve as a basis for further assignments within the heme binding site. Complete assignments, in combination with recombinant derivatives of this monomer hemoglobin, will give further insight into local interactions that influence ligand binding kinetics and heme orientational isomerism.

1H NMR Investigation of the Role of Intrinsic Heme versus Protein-Induced Rhombic Perturbations on the Electronic Structure of Low-Spin Ferrihemoproteins: Effect of Heme Substituents on Heme Orientation in Myoglobin

“…Temperature Dependence of Heme Methyl Shifts. The temperature dependence of the heme methyl shifts in 2A,4H-metMbCN and 2H,4A-metMbCN in a Curie plot (Figure 4B and D, respectively) reveals the presence of both positive (Curietype) and negative (anti-Curie-type) slopes typical of cyanometglobins, [10][11][12][13][14][15][16][17] as well as other low-spin ferric hemoproteins. 18,19 The Curie slopes and apparent interecepts in Curie plots at T -1 ) 0 for WT, 2H,4A-and 2A,4H-metMbCN are listed in Table 3.…”

“…A schematic representation of what may be expected in the simplistic picture of an imidazole aligned precisely along the N II -Fe-N III vector, strict T -1 dependence (with zero intercept) for shifts for a given orbital state, and negligible dipolar shifts (see below) is illustrated in Figure 2 for different spacing between the d xz and d yz orbitals. In fact, all cyanometglobins for which the complete hemin methyl assignments are available over a range of temperature exhibit the anomalous temperature behavior, [10][11][12][13][14][15][16] and analysis of some of these data been proposed to provide the spacing of the two relevant orbital states. 17 A similar phenomenon is observed in ferricytochromes.…”

1H NMR Characterization of Myoglobins Where Exogenous Ligands Replace the Proximal Histidine

“…For the purpose of the present study, the spectrum of reconstituted H93G(Im) after a 12 h waiting period was indistinguishable from that of protein isolated from E. coli. Absolute value COSY (MCOSY) and phase-sensitive NOESY spectra were collected following published protocols for optimizing collection of spectra for paramagnetic metcyano-heme protein complexes (Alam & Satterlee, 1994; La Mar & de Ropp, 1993; Emerson & La Mar, 1990). MCOSY spectra were collected over spectral widths of 15 000-20 000 Hz.…”

Cushioning the pressure vibration of a zeolite concentrator system using a decoupled balancing duct system

“…A clear picture of the extent of globin heterogeneity in this organism has emerged only recently, as a result of primary sequencing5, 6 and mass spectrometry 5–7. Those results have confirmed the individual uniqueness and homogeneity of the three major monomer hemoglobins, called components II, III, and IV (or alternatively GMH2, 3, and 4) that have been reproducibly isolated and characterized in this laboratory 3–8, 11–14, 24–39. Recently, the solution structure of the CO‐ligated Fe 2+ (reduced, ferrous) form of recombinant GMH4 (GMH4CO) was determined,38, 39 making it the first structure to be determined of one of our highly characterized monomer hemoglobins.…”

“…These are naturally isolated not recombinant proteins. Whereas the ferric forms are not physiologically functional, the rationale for determining these structures is that the heme paramagnetism of both unligated and CN‐ligated GMH proteins has been most useful in previous extensive NMR studies3, 4, 13, 14, 28, 29, 33, 35–37 and that cyanide binding kinetics have been measured for these proteins 31, 32. Structural and dynamics studies of native and recombinant GMH proteins are continuing, hence these structures will provide an important standard by which to compare and interpret future results.…”