Udit, Andrew K. (2005) P450 BM3 electrochemistry and electrocatalysis. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-05112005-100825
Cytochromes P450 catalyze monooxygenations of relatively inert substrates. The significance of this activity in physiology and industry has inspired many to capture this activity in vitro. However, practical applications of P450s will continue to be hindered by the need for reducing equivalents from NAD(P)H. While electrochemical methods provide a potential solution, the difficulty in achieving good electronic coupling to the heme remains an enormous obstacle.
Flavocytochrome P450 BM3 is soluble, well-characterized, and easily manipulated, making it a good target for in vitro applications. Bioelectrocatalysis was first attempted with holo BM3 using a novel electrochemical mediator, 1,1’-dicarboxycobaltocene (Mred). Absorption spectroscopy confirmed electron transfer (ET) from Mred to the cofactors, while electrolyses resulted in Mred-mediated hydroxylation of lauric acid by both the holo (16.5 nmol product / nmol enzyme / min) and heme proteins (hBM3) (1.8 nmol product / nmol enzyme / min).
Subsequent bioelectrocatalysis was attempted with the more stable hBM3. We achieved direct electrochemistry of hBM3 by wiring it through engineered surface Cys387 to a basal plane graphite electrode (BPG) with 1-pyreneiodoacetamide (Py). AFM images revealed that only pyrene-wired enzyme molecules adsorb to BPG. ko for the BPG-Py-hBM3 system was 650 ± 50/s. Rotated-disk electrode (RDE) experiments show that the BPG-Py-hBM3 system catalyzes the four-electron reduction of dioxygen to water. Analogous experiments were performed with enzyme labeled at Cys62, which is spatially adjacent to Cys387 but does not provide a similar well-coupled through-bond pathway to the heme. Surprisingly, the Cys62 mutant showed similar electrode kinetics, demonstrating that the pyrene tether does not provide a unique pathway, but probably anchors the protein onto the electrode surface in a favorable docking mode for ET.
Extensive electrochemical characterization of hBM3 was conducted in various surfactant films on BPG. Cyclic voltammetry of hBM3 in SDS films revealed the Fe(III/II) redox couple at -330 mV (vs. Ag/AgCl, pH 7.4), and ko of 40/s. Although voltammetry confirmed catalytic dioxygen reduction by Fe(II), substrate oxidation was not observed.
Voltammetry of hBM3 mutant 1-12G in DDAPSS films revealed Fe(III/II) (-202 mV) and Fe(II/I) (-1082 mV) redox couples of the heme (pH 7). Catalytic activity included dioxygen reduction by Fe(II), and reductive dehalogenation by Fe(I). Voltammetry on hBM3 in DDAPSS revealed that hBM3 and 1-12G display distinct redox properties. Absorption spectra in solution showed the Fe(III) Soret at 418 nm for hBM3, and a split Soret for 1-12G at 390 and 418 nm. Voltammetry of the proteins within DDAPSS films revealed nearly identical Fe(III/II) potentials (~ -200 mV), but significant differences in ko, 250 vs. 30 per second, and Fe(III/II) – CO potentials, -140 mV vs. -115 mV, for hBM3 vs. 1-12G. Catalytic dioxygen reduction by the proteins on RDEs was analyzed using Levich and Koutecky-Levich treatments. Calculated values of n, 2.7 vs. 4.7, and kobs, 1400000 vs. 100000/M/s, for hBM3 vs. 1-12G suggest that the two proteins differ strikingly in their reaction with dioxygen.
Using the prototypical cytochrome P450 CAM (CAM), we attempted to generate high-valent species of P450 in DDAB films. Performing rapid-scan (50 V/s) voltammetry revealed a couple (E) at 831 mV. E was not observed at scan rates less than 30 V/s at room temperature; however, at four degrees Celsius E could be reversibly generated at 1 V/s. E was found to be sensitive to imidazole in solution and to variations in pH, suggesting that the redox reaction is occurring at the metal center (i.e., Fe(IV/III)) rather than at the porphyrin macrocycle. Electrolyses revealed that the electrochemically generated high-valent species is only capable of performing S-oxidation, converting thioanisole to methyl phenyl sulfoxide.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||biocatalysis; electrochemistry; electron transfer; P450|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||9 May 2005|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||11 May 2005|
|Last Modified:||26 Dec 2012 02:41|
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