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paramagnetism of the complex, characterize the haem environment and
determine unambiguously the orientation of the cofactor in its pocket.
Because of the pseudo C 2 v symmetry of the haem group ( Fig. 6.8 ) and
the relatively small size difference between the porphyrin substituents at
positions 1 (methyl) and 4 (vinyl), and 2 (vinyl) and 3 (methyl), the haem
can sit in its protein cavity in two orientations differing approximately by
a 180 rotation about the a
g axis. The energetic difference between the
two isomers depends on the protein and can be relatively small. As a result,
many b haem proteins exhibit 'haem orientational disorder' and exist as mix-
tures ( La Mar et al., 2000 ). The two isomers have practically identical
absorption spectra, but they have distinct NMR signatures, readily identified
as such in paramagnetic states. At equilibrium, cyanomet N. commune GlbN
solutions contain two haem orientational isomers in the ratio 4:1 ( Yeh et al.,
2000 ). When gauged by the relative disposition of secondary structure ele-
ments, for example, the C-(D)-E corner, the major isomer of cyanomet
N. commune GlbN corresponds to the minor isomer of sperm whale Mb.
Other structural features are extracted from the analysis of NMR spectra.
In the cyanomet state, the chemical shift of the haemmethyl substituents can
be used to determine the orientation of the proximal histidine with respect
to the haem group ( Bertini, Luchinat, Parigi, & Walker, 1999 ). The data
Figure 6.8 The structure of b haem (iron protoporphyrin IX, protohaem) and the
nomenclature used in the text. The dashed vertical line indicates the pseudo C 2v axis
of symmetry. An axial histidine with imidazole plane oriented along this axis or in
the perpendicular direction offers the least steric clash with the porphyrin ring. The cir-
cled CD1 indicates the position of a conserved Phe in sperm whale myoglobin and
serves to orient the haem in its pocket.
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