Probing the biological significance of c‐heme attachment in cytochrome c

Abstract

Heme is an iron‐containing porphyrin ring that some iron‐dependent bacteria are able to use as their iron source. c‐type heme is differentiated from other hemes (b, d, etc.) by covalent attachment of the heme to the protein chain via two thioether bonds to Cysteines. Covalent attachment of the heme group is energetically expensive, suggesting that there is a biological benefit to the attachment. One proposed advantage, supported by our studies, is that the covalent attachment stops iron‐dependent bacteria, such as Nontypable Haemophilus influenzae (NTHi), from stealing heme. In our study, we evaluated the role of c heme attachment by creating single‐ and double‐mutant cytochrome c proteins, which lacked one or both of the covalent attachments to the heme group, respectively. We then grew NTHi in the presence of wild‐type (two covalent heme attachments), single‐mutant (one covalent attachment) and double‐mutant (no covalent attachments) protein, as well as appropriate controls, to examine the effect of covalent heme attachment on NTHi growth. Results from our studies demonstrated that the covalent linkages do prevent NTHi from scavenging heme from wild‐type and single‐mutant cytochrome c, while non‐covalently attached heme (in double‐mutant cytochrome c) is readily scavenged and used as an iron source. This study was funded by the Rochester Institute of Technology.