Environmental Engineering Reference
In-Depth Information
potentials more negative than -0.5 V at pH
7.0, and it is difficult to obtain such a
negative potential by chemical mediators or titrants, let alone provide a continuous
scale. A pyrolytic graphite 'edge' (PGE) electrode is particularly suitable: it is
easily polished by abrasion (an aqueous alumina slurry) to expose a pristine surface
and it has a very wide potential region, extending from approximately +1 V to -1 V
versus the standard hydrogen electrode (SHE) at neutral pH.
Many enzymes are now established to be excellent electrocatalysts - defined
here as catalysts for half-cell reactions that occur at an electrode [ 46 , 47 ]. Enzymes
are typically bound at the electrode by simple adsorption (exploiting a rough
electrode surface that contacts the enzyme at different points and maximizes
hydrophobic and electrostatic interactions) or by chemical attachment that may
involve engineering the enzyme to place specific residues in suitable positions for
linkage formation. It is essential that the enzyme contacts the electrode surface in
such a way that electron relay centers within the enzyme, such as the FeS clusters in
CODHs, lie within a sufficiently short distance to allow fast electron tunnelling.
For class IV CODHs, the obvious entry and exit site is the exposed D-cluster, as
emphasized in Figure 2 .
Dynamic electrochemical techniques include cyclic voltammetry (the basic
search tool, in which current is monitored as the potential is scanned) and
chronoamperometry (initiating a reaction at a fixed electrode potential, either by
stepping to that potential or injecting a reactant, and monitoring the current as a
function of time). Because a change in current corresponds directly to a change in
activity, chronoamperometry is an excellent technique for measuring how fast
inhibitors are bound or released, all under strictly controlled potential conditions.
In terms of operational advantage, PFE normally needs a relatively tiny amount
of enzyme sample, a few pmole/cm 2 - which is orders of magnitude smaller than
required for spectroscopy. Because the enzyme is immobilized on the electrode
(area typically 0.03 cm 2 ) the same sample can be successively exposed to solutions
having different pH values and inhibitor/substrate concentrations. For studying
air-sensitive enzymes such as Ni-CODH, the sealed electrochemical cell is housed
in a glove box. The electrode is normally rotated at variable high speed, which in
addition to assisting the transport of reactants to the enzyme, ensures that dispersion
of product can be controlled - the latter factor allowing product inhibition to be
investigated easily. If the substrate is a gas, this is introduced into the headspace of
the electrochemical cell and it is easy to replace substrates in the solution by
purging with inert gas.
Cyclic voltammetry provides a direct, continuous 'spectrum' of the potential
dependence. Importantly, when the enzyme catalysis approaches electrochemical
reversibility, the trace cuts across the potential axis at the equilibrium value and just
a tiny potential bias either side of this value switches the reaction between reduction
and oxidation. Such reversibility is found with many enzymes. The ratio of oxida-
tion and reduction currents measured at appropriate potential values yields the
so-called 'catalytic bias' - the tendency of the enzyme to operate preferentially in
a particular direction [ 47 ]. Overlay of scans in each direction shows that
electrocatalysis is at steady state, whereas hysteresis is a sign that the enzyme
alters its activity on a timescale that is slow relative to the rate that the potential
is scanned.
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