cheme 5.1. A The la
6.1 Abstract
The accurate measurement of a protein’s electrochemical properties is an important part of understanding its function. Several methods have been developed to facilitate communication between deeply buried protein metal centers and electrodes.
One such technique, protein film voltammetry (PFV), involves the immobilization of proteins on the surface of electrodes by various means. Such techniques can result in clear signals from proteins, allowing the measurement of not only reduction potentials but kinetics as well. Two types of PFV have been employed in the study of the nitric oxide sythase from Geobacillus stearothermophilus. First, a mutant of this NOS was covalently connected to a gold electrode. It was hypothesized that the use of a hydrophilic linker would maintain a normal aqueous environment around the enzyme and avoid the shifting of potentials (a common problem in PFV). When it was found that this technique still resulted in measuring responses with significantly shifted potentials (as compared with those measured by redox titration in solution), a more traditional film was employed. The kinetics of gsNOS was studied in DDAB films and compared with the mammalian inducible isoform. It was found to show similar behavior, and experiments are still underway to further characterize the kinetics of wild type and three mutants of gsNOS (W70H, W70F, and W70Y, introduced in Chapter 3).
6
m ea en un hy pr se st n
d v by m m as T d
.2 Introduc Key t metals. These
asily access nables the c nactivated C ydrogen fro roperties is eparates acti tandard elect o electroche
Sever eeply buried oltammetry y various me measurement methods have
s shown in T Table 6.1. T
ifferent meth
tion and Ba to the functi e metals hav s multiple o catalysis of d C-H bonds,
m water. Cl an importan ive sites from trochemical emical respon
al methods d protein m
(PFV), invo eans.4 Such t of not onl e recorded p Table 6.1 for The FeIII/II re
hods.8-10
ackground ion of most ve the unique
oxidation sta difficult but
the reductio learly, the a nt part of un m the surfac
techniques u nse can be o
have been metal centers olves the imm
techniques c ly reduction
otentials vas r the case of eduction pot
metalloenz e ability, as ates. This e t essential ch on of dinitro
ccurate mea nderstanding ce of the pro used by inor observed.
n developed s and electro
mobilization can result in n potentials stly different f a heme prot tential of a c
zymes is the distinct from electrochemi
hemical reac ogen to amm asurement of
g its functio otein by man rganic chemi
d to facilita odes. One s n of proteins clear signal but kinetics t from those tein called c cytochrome
e redox activ m most orga
ical propert ctions, e.g., monia, and t
f a protein’s on. The prot ny Ångstrom ists useless.1
ate commun such techniq s on the surf ls from prote s as well.5-7 e measured b cytochrome P
P450-BM3
vity of trans anic molecul
ty of metal the oxidatio the generatio s electrochem
tein matrix ms, renderin
1-3 In many c
nication bet que, protein face of electr eins, allowin
7 However, by other met
P450 (cyt. P4 as measure
sition es, to sites on of on of mical
often ng the cases,
tween n film
rodes ng the such thods, 450).
ed by
Discrepancies in measured reduction potentials are not limited to cyt. P450s or even iron enzymes in general. A chemically modified electrode, cysteamine on Au(111), was used to measure the CuII/I potential of the enzyme copper nitrite reductase. The authors neglect to specifically mention the measured potential in the report; however, the included cyclic voltammograms show a quasi-reversible wave slightly negative of 0 mV vs. SCE.11 The same group published later voltammetric studies of the same copper- containing nitrite reductase, this time using gold electrodes modified not with cysteamine but with self-assembled monolayers of alkane thiols.12 They again fail to mention the E1/2
they measured, but the couple clearly lies at nearly +100 mV vs. SCE. Two different monolayers on the same electrode resulted in two different reduction potentials.
Curiously, these differing potentials go undiscussed but for one mention of differing dielectric constants between films and aqueous solution.4 The cause of these shifts remains unknown.
Interestingly, varied electrochemical approaches have produced consistent results in other cases. Film voltammetric methods have been used for several small electron transfer (ET) proteins, such as cytochromes c and cupredoxins, Figure 6.1. The reduction potential cupredoxin azurin, for example, falls near +300 mV vs. NHE regardless of electrochemic method.13-15 A trend seems apparent: technique-based discrepancies in reduction potential are endemic to larger metalloproteins, but measured values tend to converge as molecular size decreases.
A
C F (B ox at ac b ch sm so un m
in A
C
Figure 6.1. D B) using pho xide synthas When ttachment ha ctive sites a een studied haracterize mall ET prot oaked in pr
nclear, to d methods have
Anoth nvolves surfa
Depictions of ospholipid-li se.
n simple Co ave been em and electrode in detail.17 the electroc teins. Monol rotein solutio
ate, what ex e provided sp her non-cov factant films
f proteins im ike films (cy oulombic int mployed in o
es. Self-ass SAMs hav chemical pro
layers of mi ons, then ri xactly the n parse insight valent metho . Characteriz
B
mmobilized o ytochrome c) teractions w rder to prom sembled mon ve been used
operties of a xed hydroxy insed, and u ature of this ts),11 but cle od of attach
zation of a p
on electrode ),4 and (C) u will not suff mote electron nolayers (SA d extensively
azurin and yl and alkyl- used in volt s interaction ear signals ca hing a prot protein samp
s (A) using using a DDA ffice, alterna nic coupling AMs) on go y by Gray a cytochrome -terminated t tammetric e n is (surface an be record tein sample ple within th
SAMs (azur AB film and ative method g between pr old surfaces and coworke e c, among
thiols on gol xpierments.
e characteriz ed.
to an elec his matrix is
rin),16 nitric ds of rotein
have ers to other ld are It is zation
ctrode s very
difficult; only when using a specially designed cell and extensive reflections can one take a UV-visible or IR spectrum of a protein within a film.18 Films have successfully been employed with a number of enzymes including cyt. P450, myoglobin, and nitric oxide synthase.19 A selection of some of the surfactants that have been used for film voltammetry is shown in Scheme 6.1.