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 αβ2 [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. & Yamamura, Y. (1966) Biochim. Biophys. Acta 127, 280–2821, 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 (αcometβ), valency hybrid or the (αcoβco), 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.

The Apolar Distal Histidine Mutant (His69→Val) of the Homodimeric Scapharca Hemoglobin Is in an R-like Conformation

“…Their increased rates may be attributed to relief of steric restraints close to the heme, coupled with partial breakdown of the histidine gate, leading to very high rates of oxygen dissociation from the sperm whale mutants. In this connection it is of interest that in βE7Phe, the only apolar mutant of HbA studied, the intrinsic ligand binding parameters are altered only slightly with respect to natural HbA since the effect of the mutation can be entirely accounted for in terms of a stabilization of the R-state (6,26). In turn, the behavior of the HbA-βE7Phe mutant is in accordance with the suggestion that the distal pocket in liganded β subunits is more open than in myoglobin (27).…”

Nonvertebrate Hemoglobins: Functions and Molecular Adaptations

“…3), which decrease drastically at low pH due to pronounced Bohr effects, indicating that the upper part of their O 2 binding curves is not exploited in life (14,94,187,259,260,383,549,649). The presence of a distal Phe observed in the sequence of a Eudistylia Chl globin chain (L. Moens, personal communication) correlates with the low O 2 affinity of the native complex since a distal Phe substitution in HbA leads to decrease in affinity (297). Studies of Eudistylia Chl structure using chemical dissociation and ESI-MS (211,441) have shown it to consist of disulfide-bonded dimers and tetramers of several globin chains, which form noncovalent tetramers and dodecamer subassemblies, respectively.…”

Phosphate group effects upon the equilibrium of iron(III) meso-tetrakis (4-N-methylpyridiniumyl) porphyrin in aqueous solution

“…It is wellknown from several recent literature works that a pentacoordinated species presents simultaneously a less intense and blue-shifted Soret band as compared to a hexacoordinated species. This behavior has been observed both in model systems based in coordination compounds [6,14] as well as in various heme proteins [15][16][17][18][19][20]. Visca and co-workers [21] argue that a Soret band from a pentacoordinated species usually presents a molar absorptivity (e) which is only 60% of that of a hexacoordinated species.…”