Using NTHi growth studies to demonstrate the biological significance of c‐heme covalent attachment

The property that defines c ‐type heme proteins is the covalent attachment of the heme group to the polypeptide chain of the protein, such as in cytochrome c . The covalent attachment of the c ‐heme (usually by thioether linkages donated by two Cysteine residues) is energetically expensive, and the biological motivation for the attachment is unknown. We propose that the biological significance of the covalently attached c ‐heme is to prevent heme‐dependent bacteria, such as Nontypable Haemophilus influenzae (NTHi), from scavenging or stealing heme from c ‐heme proteins. In our study, we prepared wild‐type, single and double mutant cytochromes c using recombinant DNA technology, protein expression in Escherichia coli , and purification via ion affinity column chromatography. The wild‐type, single, and double mutant proteins contain two, one, and zero covalent heme attachments, respectively. We then grew NTHi in the presence of all three proteins (as well as hemin and water as controls). The results of our results suggest that, indeed, cytochrome c can only act as a heme source for NTHi when one or both covalent heme attachments are removed. This study was funded by the Rochester Institute of Technology.