1) Why is it so challenging to determine how much of what type of a metal is

bound to a protein?
The data obtained by determining the type of metal, stoichiometry and the location
of metals in a particular protein, is vital in order to explain their function(s) and to
manipulate their abilities to make useful chemical compounds including catalysts in
chemistry. The challenges in determining above data mainly depend on the type of
the protein as well as the methodology that is being used. Among the many
methods X-ray diffraction (XRD) crystallography plays an important role in
characterizing these protein, especially in determining the locations and the
identities of the metals.
XRD although gives a real image of the analyzed protein it accompanies certain
challenges as mentioned below. First of all, the analytic protein should be crystalline
in order to that to be analyzed. Making a crystalline sample of a protein is easier
said than done as different proteins might get crystallized under different pH and
temperature ranges and with different solvents. Choosing an optimum pH is
important as most of the time the lower the pH the more tendency is there for
loosing metal centers from the active site while they are convenient to be
crystallized at lower pHs. An example for this would be the detection of extremely
low concentration of Fe (iii) in mouse R2 (~0.4) at pH 4.7 showing the effect of pH.
The temperature can affect in denaturing the protein or in changing the active
conformation of the protein. In addition to that these problems, metal centers can
be sensitive to the purification processes and might get washed away. Moreover,
crystalizing integral membrane bound proteins is challenging, owing to their lack of
hydrophilic or polar surfaces.
Exposure to air can affect the metal content available in the protein as well as to
the active conformation of the protein and thus can finally affect the crystal
structure. Among the many possible ways to go wrong, the presence of adventious
metals at active sites and at biologically nonspecific sites can give rise to lot of
errors in the results. An example for this would be the presence of Zn(ii) instead of
Fe(iii) in yeast R2. However it is known that absorption of X-ray by metals can give
rise to anomalous behavior of scattering owing to the breakdown of Friedels law.
With this in mind one can identify the type of metal for certainty by irradiating the
sample with the corresponding edge energy and compare the Patterson maps with
and without irradiation. Namely there are two methods which uses this concept and
those are multiwavelength anomalous dispersion (MAD) and single wavelength
anomalous dispersion (SAD) which I think are highly useful in determining the
identity of the metal. Another problem that occurs in XRD its is low resolution which
could deprive seeing the metal centers clearly and distinguishing metal centers
from metal clusters. Resolution is particularly important in identifying dimers.
Determining the stoichiometry of the metal with respect to a protein also requires
the determination of the concentration of the particular protein which is quite
ambiguous to a certain extent. For the record there are methods such as Bradford
assay, bicinchoninic acid assay and the use of the extinction coefficient at 280 nm.
However these methods may give inconsistent results to one another. This could
contribute a lot of uncertainty in results.
Above mentioned challenges and abilities of XRD paves the way to use it with other
types techniques such as EPR, EXAFS and various other spectroscopic methods
along with it. However EPR could also give misleading results as it mostly detects

only the overall spin of a metal cluster. In addition, methods such as ICP-AES and
AAS can be effective in determining the metal content accurately. All in all
determining the type and how much of a metal in a protein is quite challenging and
would require attacking the research problem in several different ways. Moreover
the results could affect the mistakes and inadequacies of the methods as well as the
uncertainty of nature by itself manifested in proteins makes it highly challenging to
analyze metalloenzymes. Yet it is highly important to science and is worth putting
an effort into it.