A nuclear overhauser study of heme orientational isomerism in monomeric Chironomus hemoglobins

The nuclear Overhauser effect (NOE) was used to investigate heme orientation and to obtain assignments for all resolved resonances in the 1H-NMR spectrum of met-cyano Chironomus thummi thummi monomeric hemoglobins III and IV (Hb III and Hb IV). The only non-heme resolved resonance was found to be from Phe-38 (CD1), and NOE dipolar connectivity between this resonance and the heme 5- and 8-methyls was used to establish the absolute orientation of the heme for each heme-insertion isomer present. The assignments of resonances and heme disorder permitted structural comparisons between the various components, including those due to a point mutation in Hb III. Finally, the characteristic differences of NOE patterns to amino-acid protons from substituents on heme pyrroles I and II formed the basis for assigning resonances and heme orientation relative to native Hb IV for deuterohemin-reconstituted Hb IV, for which there are no X-ray data available.