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Measurement of electron transfer through cytochrome P450 protein on nanopillars and the effect of bound substrates.

ABSTRACT: Electron transfer in cytochrome P450 enzymes is a fundamental process for activity. It is difficult to measure electron transfer in these enzymes because under the conditions typically used they exist in a variety of states. Using nanotechnology-based techniques, gold conducting nanopillars were constructed in an indexed array. The P450 enzyme CYP2C9 was attached to each of these nanopillars, and conductivity measurements made using conducting probe atomic force microscopy under constant force conditions. The conductivity measurements were made on CYP2C9 alone and with bound substrates, a bound substrate-effector pair, and a bound inhibitor. Fitting of the data with the Poole-Frenkel model indicates a correlation between the barrier height for electron transfer and the ease of CYP2C9-mediated metabolism of the bound substrates, though the spin state of iron is not well correlated. The approach described here should have broad application to the measurement of electron transfer in P450 enzymes and other metalloenzymes.

SUBMITTER: Jett JE

PROVIDER: S-EPMC3876957 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Measurement of electron transfer through cytochrome P450 protein on nanopillars and the effect of bound substrates.

Jett John E JE Lederman David D Wollenberg Lance A LA Li Debin D Flora Darcy R DR Bostick Christopher D CD Tracy Timothy S TS Gannett Peter M PM

Journal of the American Chemical Society 20130304 10

Electron transfer in cytochrome P450 enzymes is a fundamental process for activity. It is difficult to measure electron transfer in these enzymes because under the conditions typically used they exist in a variety of states. Using nanotechnology-based techniques, gold conducting nanopillars were constructed in an indexed array. The P450 enzyme CYP2C9 was attached to each of these nanopillars, and conductivity measurements made using conducting probe atomic force microscopy under constant force c ...[more]

PMID: 23427827

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Project description:Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in Archaea IMPORTANCE The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane.

Dataset Information

Measurement of electron transfer through cytochrome P450 protein on nanopillars and the effect of bound substrates.

Publications

Measurement of electron transfer through cytochrome P450 protein on nanopillars and the effect of bound substrates.

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