BPS2026 – Purification and characterization of the iron oxidizing porin, cytochrome Cyc2, from seafloor iron-rich microbial mats

Iron plays a key role in regulating primary productivity and carbon cycling in marine ecosystems. However, its bioavailability largely depends on the oxidation from ferrous to ferric iron which is mediated by both abiotic factors, such as oxygen and pH and biotic utilization by several microbial communities. Among these communities, Zetaproteobacteria are chemolithotrophic bacteria which gain energy from iron oxidation at circumneutral pH and low oxygen environments where competition with abiotic oxidation is reduced. Within, Ghiorsea bivora TAG-1, a genus recently isolated from iron-rich microbial mat differs from other Zetaproteobacteria in its ability to achieve chemolithoautotrophy using either hydrogen or ferrous iron. To investigate this iron oxidation system, numerous metagenomic, metatranscriptomic, and chemical assays were carried out, leading to the identification of an outer membrane protein, Cyc2, as a key protein involved in iron oxidation as well as other components that may be implicated in this system. The aim of this study is to characterize G. bivora ’ s Cyc2 to gain deeper insights into its role and better understand iron oxidation processes. For the production part, we succeeded in expressing, solubilizing, and purifying Cyc2 from E. coli . Biochemical characterization through spectroscopic analysis confirmed it as hemoprotein. In addition, by setting up an iron oxidizing activity assay, we were able to validate its ferroxidase activity and carry out kinetic studies to obtain Cyc2’s kinetic constant. Current work aims to calculate its redox potential and have insights on its structure by using nuclear magnetic resonance (NMR). Finally, in order to recreate the physiological conditions encountered on the ocean floor, we intend to study its activity at high hydrostatic pressure comparable to that at which iron rich mats are sampled.