COMPLETE HEME PROTON HYPERFINE RESONANCE ASSIGNMENTS OF THE GLYCERA-DIBRANCHIATA COMPONENT-IV METCYANO MONOMER HEMOGLOBIN

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.