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

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.