A Reduced Oxy Intermediate of Cytochrome P450(cam) Involved in Dioxygen Activation

Observation of P450 intermediates involved in hydrocarbon hydroxylation and dioxygen activation have been precluded by the reduction of the oxy intermediate being the rate determining step. D251N cytochrome P450$\sb{\rm cam}$ slowed the overall catalytic rate, while maintaining full catalytic coupling, while exhibiting a new UV-visible spectrum under catalytic turnover that was red shifted from all other catalytic intermediates (Gerber and Sligar, 1994). The kinetics of intermediate formation (UV-visible), camphor hydroxylation (GC), reduced putidaredoxin oxidation (EPR) were measured in parallel for the reaction of ferrous deoxy P450 and reduced putidaredoxin with dioxygen. This reaction initially formed oxy P450, which subsequently reacted with the reduced putidaredoxin. Within the first 25 seconds, the UV-Visible spectral intermediate had been fully formed and 1.0 equivalent of reduced putidaredoxin was oxidized, where as less than 10% 5-hydroxycamphor had been formed. One equivalent of reduced putidaredoxin was oxidized in the first 25 seconds. These results clearly demonstrate that the reduction of oxy P450 (which was the rate determining step for the wild type enzyme) was not the rate determining step in D251N P450$\sb{\rm cam}$ catalysis. Resonance Raman spectroscopy demonstrated that the additional electron density was not on the O-O bond, which indicates the intermediates is before O-O bond heterolysis but excludes the possibility of a ferric hydroperoxo intermediate. Therefore, the reduced oxy intermediate must occur before the first proton transfer. The lack of a change in the heme marker bands relative to oxy P450, by resonance Raman, confirms that the additional electron was not observed on the heme iron. Occupation of the additional electron on a sulfur based molecular orbital was consistent with various theoretical models, marked increases in the geminate recombination rate, and a new band observed at 394 cm$\sp{-1}$ in transient resonance Raman experiments. The lack of nucleophilic reactivity, along with solely peroxide branchpoint uncoupling with various substrates, is consistent with an additional electron residing on a sulfur based molecular orbital.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Benson, David Eric

Contributors dc:contributor

• Sligar, Stephen G.
• Kenneth S. Suslick

• Biophysics, General

Language dc:language

eng

Identifier

(MiAaPQ)AAI9812533

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/84374