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Electrochemical Hydroxylation of C3–C12 n-Alkanes by Recombinant Alkane Hydroxylase (AlkB) and Rubredoxin-2 (AlkG) from Pseudomonas putida GPo1

Tsai, Yi-Fang, Luo, Wen-I, Chang, Jen-Lin, Chang, Chun-Wei, Chuang, Huai-Chun, Ramu, Ravirala, Wei, Guor-Tzo, Zen, Jyh-Myng, Yu, Steve S.-F.·2017·Scientific Reports
DOI 10.1038/s41598-017-08610-wPMC PMC5566439
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Abstract

An unprecedented method for the efficient conversion of C 3–C 12 linear alkanes to their corresponding primary alcohols mediated by the membrane-bound alkane hydroxylase (AlkB) from Pseudomonas putida GPo1 is demonstrated. The X-ray absorption spectroscopy (XAS) studies support that electrons can be transferred from the reduced AlkG (rubredoxin-2, the redox partner of AlkB) to AlkB in a two-phase manner. Based on this observation, an approach for the electrocatalytic conversion from alkanes to alcohols mediated by AlkB using an AlkG immobilized screen-printed carbon electrode (SPCE) is developed. The framework distortion of AlkB–AlkG adduct on SPCE surface might create promiscuity toward gaseous substrates. Hence, small alkanes including propane and n-butane can be accommodated in the hydrophobic pocket of AlkB for C–H bond activation. The proof of concept herein advances the development of artificial C–H bond activation catalysts.

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Figure 1 Electron pathway and protein components involved in the catalytic oxidation of medium-chain length n-alkane. The oxidation is mediated by AlkB with electrons supplied from AlkG.
Figure 1 Electron pathway and protein components involved in the catalytic oxidation of medium-chain length n-alkane. The oxidation is mediated by AlkB with electrons supplied from AlkG.CC BY · PubMed Central
Figure 2 (a) Normalized XANES spectra of FeSO4 (light green), FeCl3 (cyan), AlkGreduced (magenta), AlkGoxidized (blue), AlkBreduced (red) and AlkBoxidized (black). The inset highlights the pre-edge absorption of AlkBoxidized, AlkGoxidized and AlkGreduced at ca. 7113 eV; (b) Titration of dithionite into the mixture of AlkG and AlkB with ca. 1:1 ratio (direction for the titration of dithionite, concentration from low to high: Black → Red → Blue).
Figure 2 (a) Normalized XANES spectra of FeSO4 (light green), FeCl3 (cyan), AlkGreduced (magenta), AlkGoxidized (blue), AlkBreduced (red) and AlkBoxidized (black). The inset highlights the pre-edge absorption of AlkBoxidized, AlkGoxidized and AlkGreduced at ca. 7113 eV; (b) Titration of dithionite into the mixture of AlkG and AlkB with ca. 1:1 ratio (direction for the titration of dithionite, concentration from low to high: Black → Red → Blue).CC BY · PubMed Central
Figure 3 (a) Pre-edge spectra of AlkGreduced (magenta), AlkGoxidized (blue) and AlkBoxidized (black) after the subtraction from background; (b) the pre-edge spectrum of AlkBoxidized (red circled line) is de-convoluted into two pre-edge peak components (dashed lines) with the absorption maximum at 7114.1 eV and 7115.9 eV, respectively. The merged spectra of two component peaks (black line, 11.2 units) and the pre-edge spectrum of AlkBoxidized are superimposable.
Figure 3 (a) Pre-edge spectra of AlkGreduced (magenta), AlkGoxidized (blue) and AlkBoxidized (black) after the subtraction from background; (b) the pre-edge spectrum of AlkBoxidized (red circled line) is de-convoluted into two pre-edge peak components (dashed lines) with the absorption maximum at 7114.1 eV and 7115.9 eV, respectively. The merged spectra of two component peaks (black line, 11.2 units) and the pre-edge spectrum of AlkBoxidized are superimposable.CC BY · PubMed Central
Figure 4 Left: Phase shift corrected Fourier transforms of Fe EXAFS (red circles) and the corresponding best fits (black solid lines); Right: Fe EXAFS (red circles) and the corresponding best fits (black solid lines) of purified AlkB.
Figure 4 Left: Phase shift corrected Fourier transforms of Fe EXAFS (red circles) and the corresponding best fits (black solid lines); Right: Fe EXAFS (red circles) and the corresponding best fits (black solid lines) of purified AlkB.CC BY · PubMed Central
Figure 5 (a) Electrochemical impedance studies of AlkG modified SPCE, AlkB-enriched membrane SPCE, and bare SPCE in 1 mM [Fe(CN)6]3−/0.1 M KCl solution. Electrochemical studies in 0.1 M phosphate buffer solution (PBS), pH 7.4: (b) cyclic voltammograms (CVs) of bare SPCE measured in the absence (black dotted line) and in the presence (black solid line) of oxygen; and CVs of AlkG-SPCE measured in the absence (red dash line) and in the presence (blue solid line) of oxygen. Inset: DET of AlkG-SPCE obtained in the absence of oxygen. (c) CVs of bare SPCE measured in the absence (black dotted line) and in the presence (black solid line) of oxygen; and CVs of AlkB-enriched membrane SPCE in the absence (red dash line) and in the presence (blue solid line) of oxygen. Inset: DET of AlkB-enriched membrane SPCE in the absence of oxygen.
Figure 5 (a) Electrochemical impedance studies of AlkG modified SPCE, AlkB-enriched membrane SPCE, and bare SPCE in 1 mM [Fe(CN)6]3−/0.1 M KCl solution. Electrochemical studies in 0.1 M phosphate buffer solution (PBS), pH 7.4: (b) cyclic voltammograms (CVs) of bare SPCE measured in the absence (black dotted line) and in the presence (black solid line) of oxygen; and CVs of AlkG-SPCE measured in the absence (red dash line) and in the presence (blue solid line) of oxygen. Inset: DET of AlkG-SPCE obtained in the absence of oxygen. (c) CVs of bare SPCE measured in the absence (black dotted line) and in the presence (black solid line) of oxygen; and CVs of AlkB-enriched membrane SPCE in the absence (red dash line) and in the presence (blue solid line) of oxygen. Inset: DET of AlkB-enriched membrane SPCE in the absence of oxygen.CC BY · PubMed Central
Figure 6 Specific activity in turnover frequency (TOF) with increasing concentration of AlkB bound to AlkG for the catalytic conversion of n-octane to 1-octanol. The red curve is resulted from the fitting using an apparent two-site binding model.
Figure 6 Specific activity in turnover frequency (TOF) with increasing concentration of AlkB bound to AlkG for the catalytic conversion of n-octane to 1-octanol. The red curve is resulted from the fitting using an apparent two-site binding model.CC BY · PubMed Central
Figure 7 Specific activity in turnover frequency (TOF) for the electrochemical conversion of medium-chain length n-alkanes (C3–C12) to primary alcohols mediated by AlkB-enriched membrane bound to AlkG immobilized on a SPCE.
Figure 7 Specific activity in turnover frequency (TOF) for the electrochemical conversion of medium-chain length n-alkanes (C3–C12) to primary alcohols mediated by AlkB-enriched membrane bound to AlkG immobilized on a SPCE.CC BY · PubMed Central

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Authors
Tsai, Y.-F., Luo, W.-I., Chang, J.-L., Chang, C.-W., Chuang, H.-C., Ramu, R., Wei, G.-T., Zen, J.-M., & Yu, S. S.-F.
ORCID
Yu, Steve S.-F.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-08-21
Volume
7
Issue
1
ISSN
2045-2322
DOI
10.1038/s41598-017-08610-w
PMC
PMC5566439
Cited by
31
References
0
Language
en
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