Characterization of the key membrane protein cytochrome Cyc2 from Ghiorsea bivora

This study reports the heterologous production, purification, and characterization of the cytochrome Cyc2_TAG1 cluster 1 (Cyc2_{TAG1_1}) from Ghiorsea bivora TAG1 in the outer membrane of Escherichia coli BL21. Using a specific gene construct we optimized solubilization and purification protocols to obtain high-purity enzyme. This enabled biophysical characterization by EPR, measurement of its midpoint redox potential, and the development of an iron-oxidizing assay with 2,6-dichlorophenolindophenol (DCPIP) as an artificial final electron acceptor. The study provides the first quantitative kinetic characterization of a Cyc2 homolog in vitro. The absence of comparable apparent K_m or V_max values with a natural or artificial electron acceptor in the literature underscores the novelty of our findings. Cyc2_{TAG1_1} exhibited an apparent K_m of 627 ± 226 μm for iron sulfate, consistent with iron-rich hydrothermal vent environments where G. bivora thrives, and an apparent V_max of 19 ± 5 μm·s^-1. Despite using an artificial electron acceptor that does not accurately represent the natural partner or the real energetic system, the enzyme exhibits apparent kinetic parameters that retain biological significance, with a catalytic efficiency within the range expected for functional electron transfer enzymes. Cyc2_{TAG1_1} exhibited a redox potential of -130 ± 20 mV vs SHE which is the lowest reported for Cyc2 homologs. The EPR spectrum is consistent with a low-spin ferric heme with two axial ligands within the protein barrel. These findings provide the first biochemical characterization of Cyc2_{TAG1_1} and support its proposed role in iron oxidation.