An Active and Versatile Electron Transport System for Cytochrome P450 Monooxygenases from the Alkane Degrading Organism Acinetobacter sp. OC4.

Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobacter sp. OC4 are investigated. Upon heterologous production of both proteins independently in Escherichia coli, spectral analysis showed their reduction capability towards reporter electron acceptors, e. g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25 U mg-1. Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK]n, flexible glycine linkers [GGGGS]n or rigid proline linkers [EPPPP]n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19 U mg-1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily.

Correction: The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal.

[This corrects the article DOI: 10.1039/D2SC00385F.].

The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal.

[FeFe]-hydrogenases catalyze the reversible conversion of molecular hydrogen into protons and electrons with remarkable efficiency. However, their industrial applications are limited by their oxygen sensitivity. Recently, it was shown that the [FeFe]-hydrogenase from Clostridium beijerinckii (CbA5H) is oxygen-resistant and can be reactivated after oxygen exposure. In this work, we used multifrequency continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy to characterize the active center of CbA5H, the H-cluster. Under oxidizing conditions, the spectra were dominated by an additional and unprecedented radical species. The generation of this radical signal depends on the presence of an intact H-cluster and a complete proton transfer pathway including the bridging azadithiolate ligand. Selective 57Fe enrichment combined with isotope-sensitive electron-nuclear double resonance (ENDOR) spectroscopy revealed a spin density distribution that resembles an H-cluster state. Overall, we uncovered a radical species in CbA5H that is potentially involved in the redox sensing of CbA5H.

Fine-tuning of FeS proteins monitored via pulsed EPR redox potentiometry at Q-band.

As essential electron translocating proteins in photosynthetic organisms, multiple plant-type ferredoxin (Fdx) isoforms are involved in a high number of reductive metabolic processes in the chloroplast. To allow quick cellular responses under changing environmental conditions, different plant-type Fdxs in Chlamydomonas reinhardtii were suggested to have adapted their midpoint potentials to a wide range of interaction partners. We performed pulsed electron paramagnetic resonance (EPR) monitored redox potentiometry at Q-band on three Fdx isoforms for a straightforward determination of their midpoint potentials. Additionally, site-directed mutagenesis was used to tune the midpoint potential of CrFdx1 in a range of approximately -338 to -511 mV, confirming the importance of single positions in the protein environment surrounding the [2Fe2S] cluster. Our results present a new target for future studies aiming to modify the catalytic activity of CrFdx1 that plays an essential role either as electron acceptor of photosystem I or as electron donor to hydrogenases under certain conditions. Additionally, the precisely determined redox potentials in this work using pulsed EPR demonstrate an alternative method that provides additional advantages compared with the well-established continuous wave EPR technique.