CHARACTERIZATION OF HEME OXYGENASE AND FERREDOXIN NADPH REDUCTASE: TWO REDOX PARTNER PROTEINS INVOLVED IN THE HEME-IRON ACQUISITION PATHWAY OF PSEUDOMONAS AERUGINOSA

Abstract

The infections from bacterial pathogen Pseudomonas aerugionsa are very difficult to cure and cause premature death of many patients with AIDs, cystic fibrosis and cancer. Bacteria need iron to infect an organism, but the concentration of free iron in mammals is very low (~10-9 M). Therefore, bacteria have developed sophisticated iron acquisition mechanisms, including the utilization of heme iron. Heme oxygenase is the only known protein used for heme degradation and iron release inside a cell. In Pseudomonas aerugionsa, once heme is internalized to the cytoplasm, heme oxygenase (pa-HO) uses heme as a cofactor and substrate and degrades it into biliverdin and releases iron and CO. This heme degration process uses three oxygen molecules and seven electrons obtained from a novel redox partner protein: ferredoxin NADPH reductase (pa-FPR). The unique heme degradation catalysis in pa-HO was studied from a global view by probing a series of hydrophobic phenylalanine (Phe) rings inside heme oxygenase. We probed the Phe rings via mutagenesis, fluorine labeling using reactivity assays and spectroscopic methods to understand the relationship of protein structure, dynamics and catalysis. The results show that the hydrophobic cluster not only maintains protein structure, but also fine-tunes protein dynamics to facilitate the channeled H-bonding network from the heme binding site to the protein periphery to sustain catalysis.