Peroxo-iron and Oxenoid-iron Species as Alternative Oxygenating Agents in Cytochrome P450-catalyzed Reactions: Switching by Threonine-302 to Alanine Mutagenesis of Cytochrome P450 2B4.

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Gregory M. Raner, Associate Professor and Graduate Director (Creator)
The University of North Carolina at Greensboro (UNCG )
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Abstract: ABSTRACT Among biological catalysts, cytochrome P450 is unmatchedi n its multiplicityo f isoforms,i nducers, substrates,a nd typeso f chemical reactionsc atalyzed.I n the presents tudy,e videncei s givent hatt hisv ersatilityex tendst o the nature of the active oxidant. Althoughm echanistice vidence froms everal laboratoriesp ointst o a hypervalentir onoxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar,M ., Calder, M. R., Corina, D. L. & WrightJ, . N. (1982) BiochemJ. . 201,5 69-580] proposed that in steroidd eformylatioenf fectedb y P450 aromatase an iron-peroxos pecies is involvedW. e have shownm orer ecently that purifiedl iver microsomalP 450 cytochromesi,n cluding phenobarbital-inducedP 450 2B4, catalyze the analogous deformylationo f a series of xenobiotica ldehydes with olefin formationT. he investigationp resentedh ere on the effecto f site-directedm utagenesiso f threonine-302t o alanine on the activitieso f recombinantP 450 2B4 with N-terminala mino acids 2-27 deleted [2B4 (A2-27)] makes use of evidencef rom otherl aboratoriest hat the correspondingm utationi n bacterial P450s interferesw ith the activationo f dioxygent o the oxenoid species by blockingp rotond eliveryt o the active site. The rates of NADPH oxidation,h ydrogenp eroxide production, a nd productf ormationf romf ours ubstrates,i ncluding formaldehydef rom benzphetamineN -demethylationa,c etophenone from1 -phenylethanoolx idation,c yclohexanolf rom cyclohexaneh ydroxylationan, d cyclohexenef romc yclohexane carboxaldehyded eformylationw, ere determinedw ith P450s 2B4, 2B4 (A2-27), and 2B4 (A2-27) T302A. Replacement of the threoniner esidue in the truncatedc ytochromge ave a 1.6- to 2.5-foldi ncrease in peroxidef ormationin the presenceo fa substrate,b ut resultedi n decreased productf ormationf rom benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold).I n sharp contrast,t hed eformylatioonf c yclohexane carboxaldehydeb y the T302A mutantw as increased about 10-foldO. n the basis oft hesef indingsa nd our previous evidencet hat aldehyded eformylationis supportedb y added H202, but not by artificial oxidants, we conclude that the iron-peroxys pecies is the directo xygend onor. It remains to be established which of the many other oxidative reactions involvingP 450 utilize this species and the extentt o which peroxo-irona nd oxenoid-ironf unctiona s alternativeo xygenating agents with the numerous isoforms of this versatile catalyst.

Additional Information

Language: English
Date: 1996
biological catalysts, peroxo-iron, oxenoid-iron, cytochrome P450, biochemistry

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