Recombinant [Pheβ63]hemoglobin shows rapid oxidation of the β chains and low-affinity, non-cooperative oxygen binding to the α subunits

Abstract

We have engineered α2β2 [Phe63]hemoglobin by changing the highly conserved distal histidine of the β chains to a phenylalanine. 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 binds only two low-affinity ligands, oxygen and azide. The absorption spectrum of the ferrous deoxy or ferric forms are not normal, displaying an enhanced absorption of the visible band near 560 nm. Half of the autooxidation process, attributed to the mutated β subunits, is over 1000-fold faster than for Hb A. The mutant Hb exhibits non-cooperative binding of two oxygens with an affinity about fivefold lower than those of HbA valency hybrids (αmetβ)2. Functional properties of this mutant Hb resemble those of Hb Saskatoon ([Tyr63]Hb) [Suzuki, T., Hayashi, A., Shimizu, A. and Yamamura, Y. (1966) Biochim. Biophys. Acta 127, 280-282]. Flash-photolysis experiments also indicate non-cooperative behaviour: the CO-recombination kinetics were independent of the fraction dissociated. Furthermore, the amplitude of the CO bimolecular phase was the same for the (α(CO)metβ)2 valency hybrid or the (α(CO)β(CO))2 form, suggesting mainly geminate CO-recombination kinetics to the β chains. EPR and Resonance Raman spectra did not show evidence for a hemichrome, normally considered as a six-coordinated iron with low-spin character. The EPR and resonance Raman spectra for the mutated β subunits demonstrate the presence of a high-spin compound in the ferric and deoxy ferrous forms. In particular, the ferrous mutated β subunits are penta-coordinated The abnormal absorption spectra are possibly due to an interaction between the porphyrin and the phenyl ring in the distal position rather than to direct binding to the iron.