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 p roteins found in the erythrocytes of the marine annelid Glycera dibran chiata. Unlike myoglobin, all three of these monomer hemoglobin compon ents lack the distal histidine, which is replaced by leucine. This sub stitution alters the protein's functional properties due to its proxim ity to the heme ligand binding site. As the initial step toward a full NMR characterization of this protein, a complete set of self-consiste nt proton NMR assignments for the heme and the proximal histidine of t he paramagnetic, metcyano form of native component IV (metGMH4CN) is p resented. These assignments relied upon a combination of one- and two- dimensional NMR spectroscopy, including nonselective spin-lattice rela xation time measurements. The metcyano form has been chosen for severa l reasons: (1) The heme paramagnetism acts as an intrinsic shift reage nt 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, ther eby allowing two-dimensional NMR experiments to be carried out at very rapid repetition rates (i.e., 5 s-1). (3) The heme proton hyperfine r esonance pattern for this paramagnetic form of wild-type monomer hemog lobin component IV provides an analytical reference for the integrity of the heme active site. This is anticipated to facilitate rapid analy sis 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 importan t, reduced form of the protein, so that the heme and proximal histidin e proton assignments will serve as a basis for further assignments wit hin the heme binding site. Complete assignments, in combination with r ecombinant derivatives of this monomer hemoglobin, will give further i nsight into local interactions that influence ligand binding kinetics and heme orientational isomerism.