MYOGLOBIN AND HEMOGLOBIN 353
- Isolation and purifi cation of the metalloprotein. For instance, Hb was fi rst
crystallized in 1849, its physiological purpose of oxygen transport was
recognized by 1864, and its molecular weight and primary amino acid
sequence was known by 1930. - Measurement of physical and spectroscopic properties of the active site.
X - ray crystallography has been extensively used to characterize metal-
loproteins, especially with regard to their metal cofactors. Interpretation
of the X - ray data must be consistent with spectroscopic data from
EXAFS, EPR, and NMR studies. Currently, X - ray crystallographic data
are deposited with the Protein Data Bank (PDB) for met - , deoxy - , and
oxyhemoglobin and myoglobin from a number of species. More recently,
X - ray studies of myoglobin and hemoglobin modifi ed by site - directed
mutagenesis of aa residues near and at the active site have led to more
detailed information on structure – function relationships. - Characterization of structural, spectroscopic, and reactivity properties of
model compounds — that is, metal cofactor small - molecule analogs. - Comparisons between the protein and the analogs to reveal new
structure – function relationships.
Chemists fi nd many advantages in studying small molecule analogs of
metalloproteins. For hemoglobin, as for other systems, one can systematically
change one variable at a time. These variables might include modifi cations of
axial ligation, binding site polarity, steric restraint, and solvent effects, among
others. The disadvantage for all small - molecule analogs is that the protective
environment of the protein itself is lost. In the heme example under discussion,
one must synthesize a complex porphyrin ligand system, keep iron in a fi ve -
coordinate state until dioxygen is added, prevent irreversible oxidation of the
iron center, and produce an environment in which O 2 ligation is favored over
CO binding.
7.2.6 Iron - Containing Model Compounds
In the case of iron - containing small molecule analogs of Mb and Hb, a rocky
road to successful model compounds was encountered. Even though the syn-
theses of iron porphyrin complexes were carried out, their irreversible oxida-
tion to the μ - oxo dimer upon addition of O 2 remained a stumbling block to
their study as small molecule analogs of Mb and Hb. Addition of dioxygen to
simple, undecorated iron porphyrins led to irreversible oxidation and into a
thermodynamic Fe(III) pit with formation of the μ - oxo dimer. This behavior
is illustrated in the following reactions adapted from reference 20 and the
scheme adapted from Suslick ’ s article as shown in Figure 7.8.^19 Initially attempts
were made to solve this problem for solution studies through the use of low
temperatures and aprotic solvents although the experimental conditions were
far from those of physiological systems.