The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.

Co-crystals of 4-hydroxybenzoic acid and 2,3,5,6-tetramethylpyrazine (2 : 1) exhibit the first supramolecular synthon polymorphism in a co-crystal; metastable anti-hierarchic polymorph I converts to stable hierarchic form II.

Comprehensive 1H NMR assignments of the heme cavity proton resonances of sperm whale metmyoglobin cyanide have provided the dipolar shifts for nonligated residues which, together with the crystal coordinates of carbonyl myoglobin, allow accurate determination of both the anisotropies and orientation of the paramagnetic susceptibility tensor, χ, in the molecular framework. The resulting axial, Δχax = 2.48 × 10-8 m3/mol, and rhombic anisotropy, Δχrh = −0.58 × 10-8 m3/mol, values at 25 °C determined from the most complete set of dipolar shifts are determined to 2% and 6% uncertainty, respectively, and agree well with theoretical estimates (Horrocks, W. D., Jr. and Greenberg, E. S. Mol. Phys. 1974, 27, 993−999). Numerically and spatially restricted input data sets lead to larger uncertainties in Δχax and Δχrh, but do not systematically bias the orientation of the tensor. Determination of the anisotropies and orientation over the temperature range 5−50 °C shows that the susceptibility tensor orientation is minimally influenced, with both anisotropies well-behaved, and with Δχax exhibiting a temperature behavior close to that predicted for the system. The quantitative determination of the magnetic anisotropies over temperature allows the quantitative separation of contact and dipolar shifts for the iron ligands. The heme contact shifts reflect the expected dominant π spin density at pyrrole positions, but the meso-protons exhibit low-field contact shifts indicative of unpaired spin in a σ orbital. Such delocalized σ spin density could arise from either deformation of the heme from planarity or the loss of σ/π separation for the d xz , d yz orbitals when the major magnetic axis is tilted strongly from the heme normal as is experimentally observed. The observed anomalous temperature dependencies of the heme methyl and axial His ring contact shifts, as well as that of the rhombic anisotropy, are all consistent with thermal population of the excited orbital state. The limitations for quanitatively determining the excited orbital state energy separation from the available NMR data are discussed.

The bivalve mollusc Lucina pectinata harbors sulfideoxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O 2 , but extraordinarily high sulfide affinity. The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86 -89). Engineering these three residues into sperm whale myoglobin results in a triple mutant with ϳ700-fold higher sulfide affinity than for wild-type. The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1 H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities. The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln 64 (E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe 68 (E11) is rotated ϳ90°about 2 and located ϳ1-2 Å closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI.

No abstract

Gastro-intestinal function plays a vital role in conditions ranging from inflammatory bowel disease and HIV through to sepsis and malnutrition. However, the techniques that are currently used to assess gut function are either highly invasive or unreliable. Here we present an alternative, non-invasive sensing modality for assessment of gut function based on fluorescence spectroscopy. In this approach, patients receive an oral dose of a fluorescent contrast agent and a fibre-optic probe is used to make fluorescence measurements through the skin. This provides a readout of the degree to which fluorescent dyes have permeated from the gut into the blood stream. We present preliminary results from our first measurements in human volunteers demonstrating the potential of the technique for non-invasive monitoring of multiple aspects of gastro-intestinal health.

No abstract

The 'H NMR hyperfine shift pattern for nonheme resonances in low-spin (S = l/2) cyanide-inhibited horseradish peroxidase, HRP-CN, and cytochrome c peroxidase, CcP-CN, are interpreted in terms of the anisotropy and orientation of the paramagnetic susceptibility tensor in the molecular framework. The orientation of the axes is obtained from a least-squares search for the Euler angles which will rotate the metal-centered crystal coordinates to a coordinate system (the magnetic axes) which quantitatively account for the observed dipolar shifts. Previous 2D NMR has shown that the key active site residues in HRP-CN (Chen, 2.; de Ropp, J. S.; Hernandez, G.; La Mar, G. N. J. Am. Chem. SOC. 1994, 116, 8772) occupy essentially the same position in the active site as in the crystal structure of CcP-CN, such that the crystal coordinates for select protons of CcP-CN could be used for both HRP-CN and CcP-CN NMR analysis. The validity of the magnetic axes description for HRP-CN is supported by the excellent correlation between observed and predicted dipolar shifts, by the ability to predict the shifts for residues not conserved between CcP and HRP, by the quantitative prediction of the influence of substrate binding on the hyperfine shifted pattern in HRP-CN, and by accounting for the significant variation in hyperfine shift pattern among cyanide-inhibited heme peroxidases genetic variants which possess very similar molecular structure. The major magnetic axis is found tilted (-20") from the heme normal in both CcP-CN and HRP-CN, and is similar to that of the Fe-CN tilt in the CcP-CN crystal structure. The effect of binding the substrate benzhydroxaminic acid to HRP-CN is to decrease the tilt of the Fe-CN unit by 9" away from the BHA binding site. The distinct patterns of hyperfine shifted for the cyanide-inhibited lignin and manganese peroxidases indicate that these proteins exhibit significantly less tilt (-5") of the Fe-CN unit from the heme normal than CcP-CN or HRP-CN.