Single molecule activity measurements of cytochrome P450 oxidoreductase reveal the existence of two discrete functional states
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Single molecule activity measurements of cytochrome P450 oxidoreductase reveal the existence of two discrete functional states. / Laursen, Tomas; Singha, Aparajita; Rantzau, Nicolai; Tutkus, Marijonas; Borch-Jensen, Jonas; Hedegård, Per; Stamou, Dimitrios; Møller, Birger Lindberg; Hatzakis, Nikos.
In: ACS chemical biology, Vol. 9, No. 3, 2014, p. 630-634.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Single molecule activity measurements of cytochrome P450 oxidoreductase reveal the existence of two discrete functional states
AU - Laursen, Tomas
AU - Singha, Aparajita
AU - Rantzau, Nicolai
AU - Tutkus, Marijonas
AU - Borch-Jensen, Jonas
AU - Hedegård, Per
AU - Stamou, Dimitrios
AU - Møller, Birger Lindberg
AU - Hatzakis, Nikos
PY - 2014
Y1 - 2014
N2 - Electron transfer between membrane spanning oxi-doreductase enzymes crucially controls vital meta-bolic processes. Here we studied for the first time with single molecule resolution the function of P450 oxidoreductase (POR), the canonical membrane spanning activator of all microsomal cytochrome P450 enzymes. Measurements and statistical analy-sis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date remained masked in bulk kinetics. To ensure that we measured the inherent behavior of POR we reconstituted the full length POR in "native like" membrane patches, nanodiscs. Nanodisc reconstitution increased stabil-ity by ~2 fold as compared to detergent solubilized POR and showed significantly increased activity at biologically relevant ionic strength conditions high-lighting the importance of studying POR function in membrane environment. This assay paves the way for studying the function of additional membrane spanning oxidoreductases with single molecule reso-lution.
AB - Electron transfer between membrane spanning oxi-doreductase enzymes crucially controls vital meta-bolic processes. Here we studied for the first time with single molecule resolution the function of P450 oxidoreductase (POR), the canonical membrane spanning activator of all microsomal cytochrome P450 enzymes. Measurements and statistical analy-sis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date remained masked in bulk kinetics. To ensure that we measured the inherent behavior of POR we reconstituted the full length POR in "native like" membrane patches, nanodiscs. Nanodisc reconstitution increased stabil-ity by ~2 fold as compared to detergent solubilized POR and showed significantly increased activity at biologically relevant ionic strength conditions high-lighting the importance of studying POR function in membrane environment. This assay paves the way for studying the function of additional membrane spanning oxidoreductases with single molecule reso-lution.
U2 - 10.1021/cb400708v
DO - 10.1021/cb400708v
M3 - Journal article
C2 - 24359083
VL - 9
SP - 630
EP - 634
JO - A C S Chemical Biology
JF - A C S Chemical Biology
SN - 1554-8929
IS - 3
ER -
ID: 95166978