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 μ m for iron sulfate, consistent with iron‐rich hydrothermal vent environments where G. bivora thrives, and an apparent V max of 19 ± 5 μ m ·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.