ELECTRON NUCLEAR DOUBLE-RESONANCE FROM HIGH-SPIN AND LOW-SPIN FERRIC HEMOGLOBINS AND MYOGLOBINS

In aquo, fluoride, azide, and cyanide derivatives of ferric hemoglobin and myoglobin, ENDOR signals have been observed and assigned. Hyperfine coupling constants have been obtained for axial ligand nuclei and for nitrogens and protons of the heme group. In aquo derivatives, but not fluoride, we have observed hyperfine couplings of about 6 MHz to exchangeable, heme-bound water protons. In both aquo and fluoride derivatives we have assigned nonexchangeable proton resonances to heme meso protons, and another exchangeable proton resonance has been assigned to the proton on the nitrogen of the proximal histidine. Small changes occur in ENDOR frequencies of these latter two types of protons on going from aquo to fluoride derivatives.,13C couplings of 28.64 ± 0.08 (myoglobin) and 27.33 ± 0.18 MHz (hemoglobin) were found for heme-bound 13CN-. The hyperfine couplings to heme meso protons of the cyanide derivatives decrease in the order protohemin, hemoglobin, myoglobin. In myoglobin and hemoglobin cyanide an exchangeable proton occurs that we estimate at 4.5-5.5 A distance from the iron. Nitrogen ENDOR seen in hemoglobin and myoglobin cyanide was assigned to heme nitrogens by comparison with the nitrogen ENDOR from hemoglobin that contained l5N in 50% enrichment only on the heme. Differences which were particularly large in the heme plane were noted between hemoglobin and myoglobin azides. The majority of the nitrogen EN DOR features from azide complexes were assigned to heme nitrogens. As measured at the g-value extremum normal to the heme plane in myoglobin and hemoglobin azides, we found 15N heme hyperfine couplings in the 7.0-8.5-MHz range, l4N heme hyperfine couplings in the 5.0-6.5-MHz range, and first-order heme 14N quadrupole couplings in the 0.4-0.6-MHz range. At the g-value extremum (gx) in the heme plane, l5N heme hyperfine couplings were in the 6.0-7.5-MHz range. We have also observed ENDOR from the nitrogens of carp hemoglobin azides, which exist in the R conformation at high pH and the T conformation at low pH. Changes in electronic g values have been seen between the two forms, but we have seen no change in the heme nitrogen ENDOR between the two forms. © 1979, American Chemical Society. All rights reserved.