146 INSTRUMENTAL METHODS
and the reference 49 authors believe that their faster electron transfer using
femtosecond instrumentation may be more characteristic of the true reaction
rate.
3.7.2.3 Time -Resolved Crystallography. Time - resolved crystallography
(TC) uses an intense synchrotron X - ray source and Laue data collection tech-
niques to greatly reduce crystallographic exposure times. Normal time resolu-
tion for X - ray crystallography has been in the range of seconds or tens of
seconds. TC has the potential to take snapshots of protein structural changes
on a nanosecond time scale. Consequently, multiple exposures may be taken
that capture the evolution of the crystallographic unit cell as it reacts over
time. Traditionally, crystallographers have applied several techniques to obtain
detailed structural information on reaction intermediates. The most common
approach has been to design a series of stable structures that mimic normally
short - lived intermediates. However, these structures are stable precisely
because they are not identical to the intermediates they seek to mimic, and
key interactions are usually missing. Other experimental techniques and chem-
ical intuition are called upon to supply the missing information, sometimes
with only limited success. One successful attempt to understand how the
attachment and release of carbon monoxide, and ultimately dioxygen, happens
on a molecular scale is described in Section 7.2.7. Rodgers and Spiro studied
the nanosecond dynamics of the R to T transition in hemoglobin.^51 Using
pulse - probe Raman spectroscopy, with probe excitation at 230 nm, these
workers were able to model the R – T interconversion of the hemoglobin mol-
ecule as it moved from the R state (HbCO) to the T state (Hb).
Time - resolved crystallography (TC), now has the potential to offer detailed
structural information on short - lived intermediates in macromolecular reac-
tions under near - physiological, crystalline conditions, and this aids elucidation
of the underlying molecular mechanisms. Interpretation of TC data has
been hindered, in part due to the diffi culty in extracting structural information
on intermediates from time - resolved electron density maps. Under certain
assumptions, these maps are weighted averages of the electron density maps
of the different structural species present at the experimental time points. That
is, these time - dependent electron density maps are structurally heterogeneous.
K. Moffat has published techniques for interpreting these maps and continues
to publish reviews updating progress in this quick - changing and expanding
fi eld.^52
In their 1996 Science article “ Photolysis of the Carbon Monoxide Complex
of Myoglobin: Nanosecond Time - Resolved Crystallography, ”^53 Moffat, Wulff,
and co - workers described the nanosecond time resolution of structural changes
that occur in the carbon monoxide complex of myoglobin (MbCO) at room
temperature on CO photodissociation by a nanosecond laser pulse. Myoglobin
(Mb) is the dioxygen - binding protein found in muscle. It contains the same
Fe(II)/Fe(III) heme cofactor as hemoglobin (Mb). Myoglobin has long
been used by researchers to study ligand - binding (O 2 versus CO versus NO,