Active intermediates in heme monooxygenase reactions as revealed by cryoreduction/annealing, EPR/ENDOR studies

Abstract

This review describes the use of cryoreduction/annealing EPR/ENDOR techniques for determining the active oxidizing species in reactions catalyzed by heme monooxygenases. The three candidate heme states are: ferric peroxo, ferric hydroperoxo, and Compound I intermediates. The enzymes discussed include cytochromes P450, nitric oxide synthase and heme oxygenase.Keywords cytochrome P450; nitric oxide synthase; heme oxygenase; ENDOR; EPR; cryoreduction; dioxygen activationThe heme monooxygenases are a superfamily of heme iron enzymes, mostly thiolate-ligated such as cytochromes P450 and NOS, but also the histidyl-ligated heme oxygenase (HO), that catalyze reductive activation of molecular oxygen for the insertion of single oxygen atom into a wide variety of substrates [1][2][3][4][5]. In this process the other oxygen atom is reduced to water. Heme monoxygenases are critical to many biological processes, including steroid hormone biosyntesis, synthesis of NO, drug metabolism and the detoxification of xenobiotics and found in most classes organisms including bacteria, fungi, plants, insects and mammals [1][2][3][4][5][6][7][8][9][10]. The reaction cycle of P450 has been the object of considerable research for over three decades. The classic cytochrome P450-type catalytic cycle is initiated by catalytic reduction of the resting-state heme iron (III) (1) to the ferrous state (2) by NAD(P)H-dependent reductases (Scheme 1), followed by binding of molecular oxygen to give the ferrous dioxy complex (3), which has been observed and characterized. It has long been thought that addition of the second electron to the ferrous oxy complex leads initially to a ferric peroxo intermediate (4)(Fe III -O 2 2-). In the classical scheme, protonation of the distal oxygen then forms a ferric hydroperoxo intermediate (5) (Fe III -OOH) [1,3,4]. A second protonation of the distal oxygen then leads to heterolytic O-O bond cleavage with loss of water and generation of iron(IV)oxo porphyrin π-cation radical (Fe(IV)=O P + ), known as Compound I (6), which was proposed to be the catalytically active species in substrate hydroxylation. However, it also has been proposed that either the 4 or 5 intermediates, as well as 6, can act as the active species in some enzymes and/or with specific classes of substrates [1][2][3][4][5][9][10][11][12][13][14][15]. In support of this idea, studies with some model heme systems showed that the reactivity of intermediates 4-6 can depend significantly on * Department of Chemistry, Northwestern University, 2145 Sheridan Road, Tech K148, Evanston, Illinois 60208, bmh@northwestern.edu, Phone: 847-491-3104, Fax: 847-491-7713. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be ...

Unveiling the crucial intermediates in androgen production

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