The active site of P450(cam) with its substrate, camphor.  The fifth ligand to heme iron is thiolate from cysteine in the protein (yellow).

The ubiquitous cytochrome P450 enzymes catalyze a wide range of biological oxidations including in the remarkably difficult hydroxylation of hydrocarbons.  Our research with cytochromes P450 focuses on the mechanism the hydroxylation reactions and the nature of the active oxidants.  Recent work has confirmed that two active electrophilic oxidant forms are produced in P450s, an iron-oxo species that has long been thought to be an oxidant, and a precursor to this species, either a hydroperoxy-iron intermediate or iron-complexed hydrogen peroxide.  The studies employed a highly precise GC-MS protocol using CI, and different kinetic isotope effects were found for two products formed by oxidation of the same position on a mechanistic probe.  Current work is focused on isolating the two oxidants via their kinetics.  Related work includes mechanistic studies of NO-synthase enzymes, the heme-containing enzymes that produce nitric oxide by oxidation of arginine.

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The active site of an sMMO hydroxylase enzyme.  The iron atoms are orange.

The methanotrophic bacteria use methane as a source of carbon.  The first functionalization reaction, conversion of methane to methanol, is one of the most difficult reactions effected in nature.  These oxidations are executed by methane monooxygenase (MMO) enzymes.  Soluble MMO enzymes have been crystallized, and their structures determined by X-ray crystallography.  They contain a diiron active site as shown in the picture.  The MMO reaction cycle is related to the P450 reaction cycle in that oxygen is the reduced to give the equivalent of hydrogen peroxide.  Also like P450, the mechanisms of the MMO hydroxylation reactions are disputed.  We are studying the kinetics of MMO oxidations directly via stopped-flow spectroscopy and indirectly with mechanistic probes.

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A number of radical kinetic studies are ongoing in the laboratory.  Most employ laser flash photolysis methods wherein the radical is produced by a laser flash, and radicals with UV chromophores are monitored.  The plot at the left shows a time-resolved spectrum obtained from a reaction of an enol ether radical cation.  The product contains a chromophore at 335 nm that is growing in with time.  The inset shows the formation of an "instantaneous" signal at 490 nm from a by-product radical produced by the laser flash and the growing signal at 335 nm from the product, a distonic radical cation with a diphenylalkyl radical moiety.

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Coenzyme B12-dependent enzymes catalyze radical rearrangements in nature involving a 1,2-shift of a group.  A typical example is the rearrangement of methylmalonyl-CoA to succinyl-CoA catalyzed by methylmalonyl-CoA mutase.  We are studying the mechanisms of these reactions by laser flash photolysis kinetic studies of model reactions.  A premise of our program is that the mechanisms of a wide range of rearrangements could be similar, involving polar effects in the radical reactions.  Our studies are unique in that we can produce the radicals in polar media including water.  Evaluation of the kinetics of the radical rearrangements as a function of pH will allow one to determine whether acid or base catalysis is important.  The program involves synthetic work to produce the model radical precursors, laser flash photolysis studies, computational studies of the reactions, and enzyme modeling.

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