Hypersensitive Radical Probe Studies of Chloroperoxidase-Catalyzed Hydroxylation Reactions

Abstract

The oxidation of hypersensitive radical probes by chloroperoxidase from Caldariomyces fumago (CPO) was studied in an attempt to "time" a putative radical intermediate. Oxidation of (trans-2-phenylcyclopropyl)methane, previously studied by Zaks and Dodds [Zaks, A., and Dodds, D. R. (1995) J. Am. Chem. Soc. 115, 10419-10424] was reinvestigated. Unrearranged oxidation products were found as previously reported, and control experiments demonstrated that the cyclic alcohol from oxidation at the cyclopropylcarbinyl position, while subject to further oxidation, survives CPO oxidation as detectable species. However, in contrast to the report by Zaks and Dodds, the rearranged alcohol product expected from ring opening of a cyclopropylcarbinyl radical intermediate was shown to be unstable toward the enzyme oxidation reaction. Because of this instability, two new hypersensitive radical probes, (trans-2-phenylcyclopropyl)ethane and 2-(trans-2-phenylcyclopropyl)propane, and their potential cyclic and acyclic products from oxidation at the cyclopropylcarbinyl position were synthesized and tested. Oxidation of both of these probes at the cyclopropylcarbinyl position by CPO gave unrearranged alcohol products only, but control experiments again demonstrated that the rearranged alcohol products were unstable toward CPO oxidation conditions. From the combination of the probe and control studies, the lifetime of a putative radical intermediate must be less than 3 ps. Whereas the results are consistent with an insertion mechanism for production of alcohol product, they do not exclude a very short-lived intermediate.

Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase

“…It should be noted that catalase does not catalyze O–O bond formation as this bond is already present in the peroxide substrate. Many of these enzymes can also catalyze the oxidation, albeit less efficiently, of more than one cosubstrate [24, 35, 36]. However, the proposed chlorite-derived compound I is surprisingly faithful to chlorite decomposition [22].…”

Picosecond Radical Kinetics. Fast Ring Openings of Secondary and Tertiary trans-2-Phenylcyclopropylcarbinyl Radicals

“…The calibration of ultrafast 2-arylcyclopropylcarbinyl radical clocks has permitted quantitative applications of the corresponding hydrocarbon precursors in studies of enzyme-catalyzed hydroxylation reactions in an attempt to implicate radical intermediates and to time the “oxygen rebound” step in these processes. Probe 1b has been used to study hydroxylation by a non-heme monooxygenase in cells of Pseudomonas oleovorans , reconstituted soluble methane monooxygenase (sMMO) hydroxylase from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, chloroperoxidase (CPO) from Caldariomyces fumago , , and various cytochrome P450 (P450) enzymes. − Probes 3b , 5b , and 6b 18 have also been used to study P450-catalyzed hydroxylation reactions.…”

A Self‐Sufficient Peroxide‐Driven Hydroxylation Biocatalyst

“…The fungal heme enzyme chloroperoxidase (CPO) has received much attention for its ability to catalyze the H 2 O 2 ‐driven, enantioselective epoxidation of alkenes 19. CPO does not efficiently hydroxylate unactivated CH bonds,20 which is the hallmark of P450 chemistry. Furthermore, protein engineering on CPO has been extremely limited, as a result of its lack of functional expression in bacteria or yeast.…”