Mechanism of ligand binding to Ni(II)-Fe(II) hybrid hemoglobins.

Abstract

The geminate and bimolecular binding of CO, O2 and NO to [alpha-Ni(II)]2-[beta-Fe(II)]2 and [alpha-Fe(II)]2-[beta-Ni(II)2] hybrid hemoglobins has been studied. Biomolecular reactions: At pH 6.6 and 20 degrees both hybrids bind CO at 0.15 x 10(6) M-1 s-1. Reactions with oxygen: At pH 6.6 the on rates are 4.8 and 7.5 x 10(6) M-1 S-1 for alpha- and beta-hybrids, respectively; the off rate is approximately 2 x 10(3) S-1 for both. At pH 8 the alpha-Fe shows cooperativity whereas the beta-hybrid does not. Nanosecond geminate reactions: Faster bimolecular rates correlate with larger geminate amplitudes; thus alpha-Fe hybrids have larger amplitudes, and O2 geminate amplitudes are larger than those with CO. At pH 8.50% of O2 recombines with the alpha-hybrid. With NO, nanosecond geminate recombination is observable only with the beta-hybrid. Picosecond reactions: alpha-Hybrids show picosecond recombination of O2. With NO, alpha-hybrids recombine at 30 ns-1, beta-hybrids at 0.3 ns-1. The NO picosecond rates correlate with the molecular dynamics which shows ligands leaving the beta-Fe atom early and regularly, but remaining near the alpha-Fe atom. The results may be explained by assuming an interaction between the alpha-subunits giving rise to a high-affinity faster-reacting form, whereas the beta-subunits only become fast-reacting when an R-T conformation change analogous to that of hemoglobin A takes place. A third allosteric state is postulated to explain the results.