464 IRON-CONTAINING PROTEINS AND ENZYMES
be discovered in the crystallographic electron density map — not a trivial end-
eavor for structures at medium or low resolution ( > 2.8 Å ) — or discovered by
other means.
Fortunately, there are corresponding advantages to the study of biological
systems using X - ray crystallography. Questions about metal ion location, the
coordination sphere of amino acid residues and exogenous ligands surround-
ing the metal, and the geometry about the metal center are routinely answered
through crystallography. Often, X - ray crystallography is a starting point for
protein and enzyme mechanistic and computational studies. Questions arising
from the protein ’ s X - ray structure often inspire researchers to synthesize
model compounds that may further clarify a catalytic cycle, or result in a small
molecule catalyst mimicking the biomolecule ’ s reaction.
In reference 190 , the authors describe the spectroscopic and X - ray crystal-
lographic techniques they used to determine the pMMO structure. First, EPR
and EXAFS experiments indicated a mononuclear, type 2 Cu(II) center ligated
by histidine residues and a copper - containing cluster characterized by a 2.57 Å
Cu – Cu interaction. A functional iron center was also indicated by Inductively
Coupled Plasma – Atomic Emission Spectroscopy (ICP – AES). 184b,187 ICP - AES
uses inductively coupled plasma to produce excited atoms that emit electro-
magnetic radiation at a wavelength characteristic of a particular element. The
intensity of this emission is indicative of the concentration of the element (iron
in this case) within the sample.
In X - ray crystallographic techniques, the reference 190 authors used copper
single - wavelength anomalous dispersion (SAD) to obtain phases, and then
they improved the phases through noncrystallographic symmetry averaging.
As described in Section 3.3.3 , the anomalous dispersion technique chooses a
wavelength (available using tunable synchrotron X - ray sources) that will cause
a transition of electrons of heavy atoms in the crystal. The wavelength chosen
is near the metal ’ s absorption edge. In this case, anomalous data collected at
an energy of 9400 eV located six copper sites arranged in pairs around a three-
fold axis. This was the fi rst indication that pMMO existed as a trimer. Addi-
tional data collection at 9686 eV identifi ed zinc ions, as well as confi rming the
presence of the copper ions. Multiple data sets at the iron edge did not reveal
the presence of iron ions in the pMMO crystal. The putative dinuclear copper
site cannot be identifi ed as such by the X - ray crystallographic analysis because
the EXAFS distance reported (Cu – Cu = 2.57 Å ) is shorter than the 2.8 - Å
maximum resolution of the PDB: 1YEW data.
In general, the collection and analysis of data at different wavelengths is an
important strategy for identifying metal ions and for solving the phase problem
for protein X - ray crystallographic structures. Tunable synchrotron radiation
sources allow data collection near the absorption edge of a specifi c metal ion.
Absorption of radiation by the metal ion leads to anomalous scattering, a
useful breakdown in Friedel ’ s law (which states that I ( h k l ) = I (( −h−k−l ).
This effect, detected in anomalous difference Fourier and Patterson maps, is
exploited in the multiwavelength anomalous dispersion (MAD) and single -