UNAMBIGUOUS HEME PROTON HYPERFINE RESONANCE ASSIGNMENTS OF A MONOMERIC HEMOGLOBIN FROM GLYCERA-DIBRANCHIATA FACILITATED WITH A COMPLETELY DEUTERATED PROTEIN

One of the most challenging tasks facing those who work with low-spin ferriheme proteins is assigning all of the heme proton NMR resonances. In part this stems from the relatively large resonance line widths an d rapid relaxation rates characteristic of hyperfine-shifted protons. Perhaps as important is that, depending upon the protein, approximatel y one-half of the hyperfine-shifted resonances are buried in a very de nse spectral region. Identifying single hyperfine-shifted proton reson ances in an extremely crowded spectrum imbues the resulting assignment s with a degree of uncertainty. In an attempt to simplify this procedu re, we have produced an extensively deuterated Component IV globin of Glycera dibranchiata in an expression system consisting of the E.; coi l strain BL21 (DE3) grown in a medium that was at least 98% deuterated . After purification, this globin was constituted with fully protonate d hemin to form the recombinant deuterated monomer hemoglobin, which w as subsequently studied by both 1D and 2D NMR methods. Complete deuter ation makes comprehensive heme proton assignments rapid to achieve, st raightforward, and unambiguous. All heme proton resonances are identif iable in partially relaxed 1D spectra, including those of the heme mes o protons. Moreover, our experience with this completely deuterated pr otein offers insights to many general and potential uses of completely deuterated proteins.