Chemistry Reference
In-Depth Information
FIGURE 6.3
AtxA and Ccc2 exchanging a Cu(I) ion. (From
Fragai, Luchinat and Parigi, et al., 2006
. Copyright 2006 with permission from
the American Chemical Society.)
mechanistic information by means of NMR that have no equivalent in the NMR study of diamagnetic proteins.
Indeed, the replacement of diamagnetic and NMR-silent metal ions by suitable paramagnetic metal ions can be
deliberately introduced into proteins to provide structural and mechanistic information not obtainable otherwise.
A good example is the incorporation of lanthanides in Ca
2
รพ
-binding sites of proteins.
It has become clear that proteins are not rigid objects, but that they can sample a rather wide range of
different conformations. NMR is a particularly appropriate technique to estimate the time scale of these
conformational changes, from seconds to picoseconds, providing information both on conformational
heterogeneity and on the time scale of motions associated with it. This is illustrated in
Figure 6.3
for the
transfer of a copper(I) ion from the copper chaperone Atx1 to the soluble domain of the Ccc2 ATPase, where in
the adduct between the two proteins, a copper-bridged intermediate is formed. In Atx1, the two metal-binding
cysteines move from a buried location in the copper(I)-loaded protein to become solvent-exposed after copper
release. In contrast, the structure of Ccc2a remains almost invariant upon binding of copper(I), indicating that
the metal-binding site in the apo-form of Ccc2a is more pre-organised than in ApoAtx1.
ELECTRONIC AND VIBRATIONAL SPECTROSCOPIES
Transitions between different electronic states result in absorption of energy in the ultraviolet, visible, and, for
many transition metal complexes, the near infrared region of the electro-magnetic spectrum. Spectroscopic
methods which probe these electronic transitions can, in favorable conditions, provide detailed information on the
electronic and magnetic properties of both the metal ion and its ligands.
Electronic spectra of metalloproteins find their origins in: (i) internal ligand absorption bands, such as
p/p
*
electronic transitions in porphyrins; (ii) transitions associated entirely with metal orbitals (d
d transitions);
e
(iii) charge transfer bands between the ligand and the metal, such as the S
Cu(II) charge
transfer bands seen in the optical spectra of Fe/S proteins and blue copper proteins, respectively.
Figure 6.4
a
presents the characteristic spectrum of cytochrome c, one of the electron transport haemoproteins of the mito-
chondrial electron transport chain. In the reduced form, this consists of four absorption bands, the
/
Fe(II) and S
/
g
(Soret) band
and three others, designated
a
,
b
, and
d
, while in the oxidised form the spectrum is characteristically different. The
three
a
bands of cytochromes a, b and c in beef heart mitochondria can be clearly distinguished (
Figure 6.4
b).
