Identification of Electron Transfer in the System of Ferredoxins and Ferredoxin Reductases from Mycolicibacterium smegmatis

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Abstract

Steroid-26-monooxygenases belong to the cytochrome P450 superfamily and function as part of three-component systems together with ferredoxins and ferredoxin reductases providing electron transport. The P450-dependent redox partners of the actinobacterial strain Mycolicibacterium smegmatis mc2155 were investigated. The genes encoding mycolibacterial ferredoxins (FdxD and FdxE) and ferredoxin reductases (FdrA and FprA) were overexpressed in E. coli cells. A scheme for isolation and purification of synthesized recombinant proteins using affinity chromatography was developed, resulting in electrophoretically homogeneous preparations. Spectral analysis of ferredoxin reductase showed absorption peaks characteristic of FAD-containing proteins. The reaction of cytochrome c reduction using recombinant proteins was carried out, demonstrating that FdxD, FdxE, FdrA, and FprA can act as components of electron transport from the reducing equivalents of NAD(P)H.

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About the authors

D. O. Epiktetov

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences, Federal Research Center

Author for correspondence.
Email: epiktetoff@gmail.com
Russian Federation, Pushchino, 142290

M. V. Karpov

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences, Federal Research Center

Email: epiktetoff@gmail.com
Russian Federation, Pushchino, 142290

M. V. Donova

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences, Federal Research Center

Email: epiktetoff@gmail.com
Russian Federation, Pushchino, 142290

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2. Fig. 1. Purification of proteins using Ni-NTA agarose: (a) Purification chromatograms FdxE, FdxD, FprA and FdrA. The arrows indicate the peaks of elution. The solid line is the absorption at 280 nm, the dotted line is the concentration of imidazole. (b) Electrophoregrams of protein preparations FprA, FdrA, FdxE and FdxD. M is a marker of molecular weights.

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3. Fig. 2. Analysis of the structure and activity of recombinant proteins: 3D models of FdrA (a) and FprA (b) proteins. Absorption spectra of FdrA and FprA (c): 1 — FAD, 2 — FprA, 3 — FdrA. The recovery rate of cytochrome with redox pairs at 550 nm (g): 1 — FdrA-FdxD, 2 — FprA-FdxD, 3 — FprA-FdxE, 4 — FdrA-FdxE.

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