NMR study of Galeorhinus japonicus myoglobin

Abstract

The molecular structure of the active site of myoglobin from the shark, Galeorhinusjaponicus, has been studied by 'H-NMR. Some hyperfine-shifted amino acid proton resonances in the met-cyano form of G. japonicus myoglobin have been unambiguously assigned by the combined use of various two-dimensional NMR techniques; they were compared with the corresponding resonances in Physter catodon myoglobin. The orientations of ThrElO and IleFG.5 residues relative to the heme in G. japonicus met-cyano myoglobin were semiquantitatively estimated from the analysis of their shifts using the magnetic susceptibility tensor determined by a method called MATDUHM ( Mol. Biol. 153, 117-1241. In spite of a substantial difference in shift between the corresponding amino acid proton resonances for the two proteins, the orientations of these amino acid residues relative to the heme in the active site of both myoglobins were found to be highly alike.NMR of paramagnetic hemoproteins has provided a wealth of information about the structural properties of their active sites [l-61. 'H-NMR resonances arising from heme peripheral groups and amino acid residues near the heme have been effectively used as a useful structural probe in the NMR study of hemoprotein because these resonances are resolved well outside the poorly resolved diamagnetic envelope in the chemical shift region of 0 -10 ppm and they are quite sensitive to the heme molecular environments. Additionally, the observation of the nuclear Overhauser effect (NOE) [7], even in the presence of paramagnetic relaxation, allows not only the signal assignments of hyperfine-shifted resonances [3, 8 -101 but also the determination of internal mobility of specific groups in the heme pocket [ l l -141.As far as the structural determination of the active site in hemoprotein is concerned, resonances of non-coordinated amino acid protons are of particular importance because of their extremely high sensitivity to the spatial position of the nuclei relative to the heme [I5 -171. Their hyperfine shifts are purely pseudo-contact shifts (dPJ and hence can be interpreted quantitatively in terms of the dipolar interaction between Correspondence to Y. Yamamoto,

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.…”

Co- and Counterrotation of Magnetic Axes and Axial Ligands in Low-Spin Ferriheme Systems

“…NMR spectroscopy has been used to determine the orientation of the g or χ tensor in a number of heme proteins, including cyanide-inhibited horseradish peroxidase, , several cyanometmyoglobins, − ferricytochrome b 5 , ,− and various cytochromes c . − These magnetic axis determinations have simply been reported in some cases, but recently Bertini and co-workers have shown that the orientation of the g or χ tensor and the dipolar (pseudocontact) shifts that result therefrom can be used as important additional constraints to help in the refinement of the 3D solution structure of the ferriheme protein . It therefore becomes important to confirm that the magnetic axes determined from a series of NMR experiments are reasonable, for it appears to be easy to make errors in assigning magnetic axis directions, especially with respect to the heme moiety within the protein.…”

“…Of the many investigations of the EPR spectra of low-spin d 5 complexes, particularly of ferriheme models and proteins, relatively few studies of the orientation of the g tensor with respect to the molecular frame have been reported. Among the existing reports are single-crystal EPR studies of two heme proteins − and a group of model ferrihemes, − and a number of NMR determinations, − as mentioned above. Recently we have investigated the magnetic field dependence of the intensity of the proton sum frequency peak in the ESEEM spectra of several model hemes to determine the orientation of the g tensor in glassy media. − In all but one 39 of these studies, g zz , the component of the g tensor aligned most closely with the z molecular axis of the heme center, has been found to be the largest g value, indicating that the electron configuration of the low-spin d 5 center is (d xy ) 2 (d xz ,d yz ) 3 .…”

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

“…For diamagnetic molecules, analyses of NOE rise-curveinitialslopes are theonly quantitativemeans of determining the pairwise internuclear cross-relaxation rate (aij). These types of experiments are the key to quantitating relative internuclear distances (Macura et al, 1981;Ernst et al, 1986;Neuhaus & Williamson, 1989;Yip, 1990;La Mar & de Ropp, 1993). The concept is a simple one-nearest neighbors display larger NOEs at shorter mixing times.…”