Structural and Functional Characterization of Iron-Containing Oxygenases from Pseudomonas aeruginosa
Pseudomonas aeruginosa is a prevalent Gram-negative opportunistic human pathogen responsible for many hospital-acquired infections. It is a significant source of infection in burn victims and is the predominant cause of morbidity and mortality in cystic fibrosis patients. P. aeruginosa is of considerable medical importance due to its high antibiotic resistance. Like most organisms, P. aeruginosa requires iron for survival and proliferation. As free iron is scarce within humans, with the majority of iron complexed with heme, this human pathogen expresses proteins responsible for heme uptake and breakdown. P. aeruginosa possesses two iron-regulated cytoplasmic heme proteins, PhuS and HemO. PhuS has been previously classified as merely a heme-trafficking protein, while HemO is the canonical heme oxygenase (HO) responsible for heme degradation to produce biliverdin, CO, and free iron. Determination of the crystal structures of PhuS in the apo- and heme-bound forms show unique structural differences compared to its homologue ChuS, the Escherichia coli O157:H7 HO. However, we have discovered that PhuS also has heme-degrading activity and is five times more potent than ChuS. We show that PhuS degrades heme to verdoheme and has a much lower energy barrier for this step than HemO. Thus, HemO is mainly responsible for the ring-opening reaction of verdoheme to biliverdin. We have also confirmed through kinetics analysis that the presence of PhuS and HemO together enhances heme degradation compared to HemO alone. These results combined have identified function coupling of this unique two-enzyme system that affords P. aeruginosa with more efficient heme breakdown and iron utilization. As iron is used as a cofactor for many enzymatic processes, we focused on another family of iron-containing oxygenase called the 2-oxoglutarate/Fe(II)-dependent oxygenases (2OG oxygenases). P. aeruginosa encode four putative 2OG oxygenases, of which two of them are characterized here: MicL (PA4191) and PiuC (PA4515). We show that MicL has the ability to bind 2OG and Fe(II), while PiuC has potential protein substrates in EF-Tu and keto-acid isomerase. Through in vivo P. aeruginosa growth assays, we show that MicL and PiuC are important regulators of cell growth, allowing the pathogen to survive longer under greater levels of nutrient stress.