RECOMBINANT [PHE-BETA-63]HEMOGLOBIN SHOWS RAPID OXIDATION OF THE BETA-CHAINS AND LOW-AFFINITY, NONCOOPERATIVE OXYGEN-BINDING TO THE ALPHA-SUBUNITS

We have engineered alpha(2) beta(2) [Phe63]hemoglobin by changing the highly conserved distal histidine of the beta chains to a phenylalanin e. The mutant tetramer binds four high-affinity ligands, such as CO or NO, to the ferrous form, or CN to the oxidized iron; however, it bind s only two low-affinity ligands, oxygen and azide. The absorption spec trum of the ferrous deoxy or ferric forms are not normal, displaying a n enhanced absorption of the visible band near 560 nm. Half of the aut ooxidation process, attributed to the mutated beta subunits, is over 1 000-fold faster than for Hb A. The mutant Hb exhibits non-cooperative binding of two oxygens with an affinity about fivefold lower than thos e of HbA valency hybrids (alpha met beta)(2). Functional properties of this mutant Hb resemble those of Hb Saskatoon ([Tyr63]Hb) [Suzuki, T. , Hayashi, A., Shimizu, A. & Yamamura, Y. (1966) Biochim. Biophys. Act a 127, 280-282]. Flash-photolysis experiments also indicate non-cooper ative behaviour: the CO-recombination kinetics were independent of the fraction dissociated. Furthermore, the amplitude of the CO bimolecula r phase was the same for the (alpha(CO)met beta)(2) valency hybrid or the (alpha(CO)beta(CO))(2) form, suggest ing mainly geminate CO-recomb ination kinetics to the beta chains. EPR and Resonance Raman spectra d id not show evidence for a hemichrome, normally considered as a six-co ordinated iron with low-spin character. The EPR and resonance Raman sp ectra for the mutated beta subunits demonstrate the presence of a high -spin compound in the ferric and deoxy ferrous forms. In particular, t he ferrous mutated beta subunits are penta-coordinated. The abnormal a bsorption spectra are possibly due to an interaction between the porph yrin and the phenyl ring in the distal position rather than to direct binding to the iron.