Desaturase Reactions Complicate the Use of Norcarane as a Mechanistic Probe. Unraveling the Mixture of Twenty-Plus Products Formed in Enzyme-Catalyzed Oxidations of Norcarane

Abstract

Norcarane, bicyclo[4.1.0]heptane, has been widely used as a mechanistic probe in studies of oxidations catalyzed by several iron-containing enzymes. We report here that in addition to oxygenated products, norcarane is also oxidized by iron-containing enzymes in desaturase reactions that give 2-norcarene and 3-norcarene. Furthermore, secondary products from further oxidation reactions of the norcarenes are produced in yields that are comparable to those of the minor products from oxidation of the norcarane. We studied oxidations catalyzed by a representative spectrum of iron-containing enzymes including four cytochrome P450 enzymes, CYP2B1, CYPΔ2B4, CYPΔ2E1, and CYPΔ2E1 T303A, and three diiron enzymes, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), toluene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. 2-Norcarene and 3-norcarene and their oxidation products were found in all reaction mixtures, accounting for up to half of the oxidation products in some cases. In total, more than 20 oxidation products were identified from the enzyme-catalyzed reactions of norcarane. The putative radicalderived product from oxidation of norcarane, 3-hydroxymethylcyclohexene (21), and putative cationderived product from oxidation of norcarane, cyclohept-3-enol (22), co-elute with other oxidation products on low polarity GC columns. The yields of product 21 found in this study are smaller than those previously reported for the same or similar enzymes in studies where the products from norcarene oxidations were ignored, and, therefore, the limiting values for lifetimes of radical intermediates produced in the enzyme-catalyzed oxidation reactions are shorter than previously reported.Mechanistic probes have been used for many years to reveal details about reaction mechanisms in chemistry and biology. The concept of a mechanistic probe study is that a short-lived intermediate can be revealed by a characteristic rearrangement of a probe substrate that is observed in the reaction products. Because transient species are not followed in real time, the existence of an intermediate can only be inferred, and the validity of conclusions based on the use of probes depends on a thorough understanding of potentially complex chemistry. Norcarane (1) has been applied as a probe in studies of many enzyme-catalyzed oxidation reactions. 1-10 In most of these works, small amounts of a putative radical-derived product and a putative cation-derived product were detected, providing evidence that radicals and cations were formed as intermediates to some extent in the oxidation reactions. The evidence for radical-derived products in norcarane studies and the conclusions that radical intermediates existed is essentially unique. Most probes that were used in studies of oxidizing enzymes give the same skeletal rearrangement from radical and cation intermediates, and one cannot conclude what type of transient was involved when rearranged products are found. The ev...

Rearrangement reactions catalyzed by cytochrome P450s

“…A similar value was reported for the trapping reaction with norcarane as the substrate [22], although there is disagreement as to the extent of rearranged product formation, and thus the trapping rate [23,24]. This disagreement derives from the large number of products formed and the fact that some are formed from a cationic as opposed to radical intermediate.…”

The Enigmatic P450 Decarboxylase OleT Is Capable of, but Evolved To Frustrate, Oxygen Rebound Chemistry

“…Considering the efficient incorporation of oxygen into product alcohols, we sought to time the radical recombination reaction. Radical clock substrates have been widely used for many decades to probe the reaction mechanisms of C–H oxidizing enzymes and biomimetic model systems. ,,− However, these probes have not been examined with H 2 O 2 -utilizing CYPs. The efficient oxidation of guaiacol using DA- d 19 served as a template for testing whether a similar approach could facilitate the oxidation of norcarane (bicyclo[4.1.0]­heptane) and methylphenylcyclopropane (MCP).…”

Reduction Potentials of P450 Compounds I and II: Insight into the Thermodynamics of C–H Bond Activation

“…Figure also shows D (O–H) Ferric of the ferric water-bound heme. P450s have been found to perform desaturation and carbon–carbon bond cleaving reactions, in which P450-II oxidizes the substrate a second time. − These oxidations involve either direct H-atom abstraction or a proton-coupled oxidation of the substrate radical to generate the ferric water-bound form of the enzyme. In a similar fashion, one expects the nonrebound reactivity of P450-II toward hydrocarbons to be dictated by D (O–H) Ferric .…”