FORMATION OF 2 HYDROGEN-BONDS FROM THE GLOBIN TO THE HEME-LINKED OXYGEN MOLECULE IN ASCARIS HEMOGLOBIN

We have tried to find out why Ascaris hemoglobin has such an exception ally high oxygen affinity (P-50 approximate to 0.004 mmHg; 1 mmHg = 13 3 Pa). Following Kloek et al., we have synthesized the N-terminal glob in domain of Ascaris hemoglobin in Escherichia coil [Kloek, A. P., Yan g, J., Mathews, F. S. and Goldberg, D. (1993) J. Biol. Chem. 268, 1766 9-17671]. Like Kloek et al., we found its oxygen affinity to be as hig h as that of native Ascaris hemoglobin. We thought that this high affi nity might be due to the heme-bound oxygen molecule being stabilized b y two hydrogen bonds from the globin instead of the usual one. Ascaris hemoglobin has a distal glutamine instead of the more usual histidine as one of the potential hydrogen bond donors. In addition, it contain s a tyrosine at position 10 of B helix (B10) in place of the leucine g enerally found there in vertebrate myoglobins and hemoglobins. Followi ng the discovery of Carver et al. that sperm whale myoglobin with the replacement of leucine B10 by phenylalanine has a raised oxygen affini ty, we have replaced tyrosine B10 in the N-terminal domain of Ascaris hemoglobin by either leucine or phenylalanine [Carver, T. E., Brantley , R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, H. L., Philli ps, G. N., Jr., and Olson, J. S. (1992) J. Biol. Chem. 267, 14443-1445 0]. Either of these replacements lowered the oxygen affinity about 100 -fold, to the same level of that of human cu-globin chains. These resu lts are consistent with a hydrogen bond linking the tyrosine hydroxyl to the heme-linked oxygen, with a bond energy of 2.7 kcal/mol.