The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds. It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity. We have produced mutant haemoglobins in E. coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity.

Functionally distinct conformations of HbA (human adult hemoglobin) were probed using deoxy and diliganded derivatives of symmetric Fe-Zn hybrids of HbA. To expand the range of accessible structures, different environments were utilized including solution, sol-gel encapsulation, and crystals. Further structural and functional modulation was achieved by the addition of allosteric effectors. Functional characterization included oxygen affinity measurements, CO combination rates, and geminate and bimolecular CO recombination, after photodissociation. The conformational properties were studied using visible resonance Raman spectroscopy as a probe of local tertiary structure at the iron-containing hemes and UV resonance Raman spectroscopy as a probe of elements of the globin known to be sensitive to quaternary structure. The combined results show a pattern in which there is a progression of conformational and functional properties that are consistent with a picture in which the T quaternary structure can accommodate a range of tertiary conformations (plasticity). At one end of the distribution is the equilibrium deoxy T state conformation that has the lowest ligand reactivity. At the other end of the distribution are T state conformations with higher ligand reactivity that exhibit "loosened" T state constraints within the globin including the alpha(1)beta(2) interface and reduced proximal strain at the heme.

The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, beta-globin accumulated to approximately 10% of total E. coli proteins. alpha-Globin was not expressed in the T7 system using the native cDNA. For the expression of alpha-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains. N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue. Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.