OTHER INSTRUMENTAL METHODS 147
for instance) and conformational changes accompanying ligand - binding and
relaxation. MbCO is the most stable and easily photolyzed of the three possi-
ble systems — MbCO, MbO 2 , MbNO — and is the most popular model system.
(See Section 7.2.7 for discussion of CO binding to myoglobin.) The Fe – CO
bond was broken with a 10 - ns laser pulse and X - ray data sets were collected
at different time delays between the laser fl ash and the X - ray pulse (4 ns, 1 μ s,
7.5 μ s, 50 μ s, and 1.9 ms). Although the difference maps clearly showed release
of the CO molecule from the heme, they also suggested that CO recombina-
tion in the crystal form contains a fast, geminate phase with a recombination
rate comparable with or greater than the maximum photolysis rate applied by
the laser pulse of 10^9 s − 1. This result confi rmed that it is much more diffi cult to
photolyze MbCO molecules in the crystal form than in solution. A second
prominent feature of the X - ray difference maps arose from the motion of the
iron atom out of the heme plane and toward the proximal histidine ligand. A
third feature indicated a transient “ docking site ” for the photodissociated CO;
however, well - populated docking sites indicating CO exit from the binding
pocket were not identifi ed. A number of small electron density features indi-
cated structural rearrangements of aa residues surrounding the heme, espe-
cially the residues of the E and F helices implicated by other methods in heme
and protein relaxation effects, and in iron ion displacement in or out of the
heme plane. Their data suggested complete iron displacement and heme relax-
ation occurred in < 4 ns, in agreement with other spectroscopic results.
In 2001, the same research group published the article “ Protein Conforma-
tional Relaxation and Ligand Migration in Myoglobin: A Nanosecond to Mil-
lisecond Molecular Movie from Time - Resolved Laue X - Ray Diffraction. ”^54
Using the same techniques in the same system (MbCO), the researchers pho-
todissociated the CO ligand by a 7.5 - ns laser pulse and also probed the sub-
sequent structural changes by 150 - ps or 1 - μ s X - ray pulses at 14 laser/X - ray
delay times, ranging from 1 ns to 1.9 ms. Very fast heme and protein relaxation
involving the E and F helices was evident from the data at a 1 - ns time delay.
The photodissociated CO molecules were detected at two locations: at a distal
pocket docking site and at the Xe 1 binding site in the proximal pocket. The
so - called Xe 1 site is the highest - affi nity Xe binding site that is almost fully
occupied when metMb (Mb with Fe(III) bound in the heme ligand) are equili-
brated with seven atmospheres of Xe gas. Researchers believe that the Xe 1
site identifi es a possible proximal site on the dioxygen exit pathway from
myoglobin. The population by CO of the primary, distal site peaks at a 1 - ns
time delay and decays to half the peak value in 70 ns. The secondary, proximal
docking site reaches its highest occupancy of 20% at approximately 100 ns and
has a half - life of approximately 10 μ s. At approximately 100 ns, all CO mole-
cules are accounted for within the protein, in one of these two docking sites
or bound to the iron ion in the heme. Thereafter, the CO molecules migrate
to the solvent from which they rebind to deoxymyoglobin in a bimolecular
process with a second - order rate coeffi cient of 4.5 × 10^5 M − 1 s − 1. The results
demonstrated that structural changes as small as 0.2 Å and populations of CO