358 IRON-CONTAINING PROTEINS AND ENZYMES
and Spiro studied the nanosecond dynamics of the R to T transition in hemo-
globin.^25 Using pulse - probe Raman spectroscopy, with probe excitation at 230
nanometers, these workers were able to model the R – T interconversion of the
hemoglobin molecule as it moved from the R state (HbCO) to the T state
(Hb). Under static conditions, laser excitation at 230 nm provided resonance
enhancement of vibrational Raman bands of tyrosine (tyr α 42) and tryptophan
(trpβ 37) side - chain residues that form specifi c hydrogen bonds across the α 1 β 2
interface in the T state. These H bonds are broken in the R state. When the
researchers collected data on Hb and HbCO under transient conditions, they
found a different set of behaviors at much shorter times that reached a
maximum intensity at∼ 50 ns. They believe that these differences refl ect ter-
tiary structure changes induced by loss (deligation) of the CO ligand before
the R – T interconversion. It is known that CO molecules deligate in less than
a picosecond, may recombine with the iron center in geminate fashion before
leaving the binding pocket on a∼ 50 - ns time scale, or leave the binding pocket
on the same time scale. Therefore the workers believe that the 50 - ns differ-
ences they detect accompany carbon monoxide ligands leaving the binding
pocket rather than CO deligation. In concert with CO leaving the binding
pocket, H bonding between residues on the A helix and E helix (the E helix
being intimately connected with the heme cofactor because it contains the
distal histidine, his58) is detected. Weakening of other H bonds that would
allow a shifting of the E helix toward the heme group is also found. In X - ray
crystallographic and molecular dynamics studies, it was found that the F helix
(proximal to the heme cofactor and containing the proximal histidine ligand
his87) moves as well. Taking all this evidence into account, Rodgers and Spiro
proposed a model for the R – T reaction coordinate that starts with CO deliga-
tion and involves movement of and strain in the E and F helices (on a subpi-
cosecond time scale) along with movement of the Fe ion toward the proximal
his residue. The “ scissoring ” motion of the E and F helices, illustrated in Figure
3 of reference 25 , relieves the strain between the helices and allows the CO
to leave the binding pocket at the same time.
Moffat and co - workers reported a study of MbCO using nanosecond time -
resolved crystallography in 1996.^26 Nanosecond time - resolved crystallography
of MbCO is discussed in Section 3.7.2.3 , along with a more complete discussion
of the reference 26 work. After fi ring a 10 - ns burst of laser light to break the
CO – Fe bond, the researchers produced a diffraction image of the crystal
through application of a 150 - ps X - ray pulse. They showed release of the CO
molecule, displacement of the Fe ion toward the proximal histidine, and recom-
bination of the dissociated CO in a time frame of about 100 μ s. The reference
26 researchers found that their results using time - resolved crystallography
compared well with other spectroscopic studies of HbCO, MbCO, and their
models. See Section 3.7.2.3 for more updated examples of CO dissociation
from myoglobin and hemoglobin as detected by time - resolved X - ray crystal-
lography. These include references to movie displays illustrating CO dissocia-
tion from the heme iron.