The selection of sites for histidine introduction was guided by
inspection of the CtrHb and GlbN structures (Fig. 2). A significant
proportion of CtrHb molecules contain the heme group in a “flipped”
orientation that interchanges pyrroles A and B with pyrroles D and C,
respectively [31]. These two forms of the protein are referred to as
“major” and “minor” hemerotational isomers according to their relative
population at equilibrium. The X-ray structure (PDB ID: 1DLY [31]) is
that of the major isomer. The two heme conformations effectively
double the number of vinyl geometries available for histidine addition.
Minimal modeling (amino acid replacement in 1DLY and optimization
with UCSF Chimera [47]) suggests that a histidine at position 111, the
closest equivalent to H117 in WT GlbN, can reach the heme 2-vinyl
group as positioned in the major heme rotational isomer. The favored
rotameric state of H111 points the ring away fromthe hemetoward solvent
and generates few clashes. Another potentially reactive site, facing
the 4-vinyl group of the major heme rotational isomer,was identified at
position 75 as the equivalent of H79 in L79H GlbN. At this position,
clashes are predicted regardless of histidine rotameric state. Sites 75
and 111 were chosen despite their suboptimal geometric properties
for lack of better candidates.