The role of Thr268 and Phe393 in cytochrome P450 BM3

Jonathan P Clark, Caroline S Miles, Christopher G Mowat, Malcolm D Walkinshaw, Graeme A Reid, Simon N Daff, Stephen K Chapman

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    Abstract

    In flavocytochrome P450 BM3 there are several active site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate heme reduction potential through interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (Delta E-m [WT + arachidonate (120 mu M)] = +138 mV). Substitution of Thr268 by an alanine or asparagine residue causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (Delta E-m [T268A + arachidonate (120 mu M)] = +73 mV, Delta E-m [T268N + arachidonate (120 mu M)] = +9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady-state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. In addition, substitutions of the threonine lead to an increase in the production of hydrogen peroxide in preference to hydroxylated product. These results suggest an important role for this active site threonine in substrate recognition and in maintaining an efficiently functioning enzyme. However, the dependence of the rate constants for oxy-ferrous decay on reduction potential raises some questions as to the importance of Thr268 in iron-oxo stabilisation. (c) 2005 Elsevier Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)1075-1090
    Number of pages16
    JournalJournal of Inorganic Biochemistry
    Volume100
    Issue number5-6
    DOIs
    Publication statusPublished - May 2006
    Event12th International Conference on Biological Inorganic Chemistry - Ann Arbor
    Duration: 31 Jul 20055 Aug 2005

    Keywords

    • FLAVOCYTOCHROME P-450 BM3
    • electron transfer
    • oxygen activation
    • SITE-DIRECTED MUTAGENESIS
    • cytochrome P450
    • iron-oxo stabilization
    • CONSERVED THREONINE THR243
    • BACILLUS-MEGATERIUM
    • ACTIVE-SITE
    • CRYSTAL-STRUCTURE
    • ELECTRON-TRANSFER
    • SUBSTRATE-BINDING
    • oxyferrous species
    • OXYGEN ACTIVATION
    • CATALYTIC MECHANISM

    Cite this

    Clark, J. P., Miles, C. S., Mowat, C. G., Walkinshaw, M. D., Reid, G. A., Daff, S. N., & Chapman, S. K. (2006). The role of Thr268 and Phe393 in cytochrome P450 BM3. Journal of Inorganic Biochemistry, 100(5-6), 1075-1090. https://doi.org/10.1016/j.jinorgbio.2005.11.020