Research Topics

Cytochrome c assembly (Overview)

Since 1987 our group has studied the molecular mechanisms by which the three pathways (systems I, II, III) covalently attach heme to the protein (apocytochrome c). These systems are diagrammed on the home page. We use a combination of molecular genetics and membrane protein biochemistry to elucidate, for example, how heme moves through each system and is ultimately attached. Some of the tools we employ and details on assembly are described in the research links on this page.

We are also interested in finding chemical inhibitors of these pathways, since systems I and II are used by prokaryotes, yet humans use the simple, unrelated system III. We discovered that metal (non-iron) porphyrins specifically inhibit systems I and II, and we are developing high throughput screens for the assembly pathways (to discover new inhibitors).

Heme protein biogenesis, with emphasis on the cytochrome c biosynthetic pathways.
Location and function of c-type Cytochromes

•  c-type Cytochromes are assembled at their site of function  •

Assembly of c-type cytochromes
  • Reduced apo-protein cysteine residues spontaneously ligate to heme vinyl groups via thioether bonds.
  • c-type cytochromes are synthesized at their site of action (outside the cytoplasmic membrane in bacteria).
  • Folding occurs after ligation
Genetics and Screens
Rhodobactger Capsulatus requires c-type cytochromes for photosynthetic (anaerobic) growth but not aerobic growth. Therefore is a screen for cytochrome c.
The classic “oxidase test” is a secondary screen
Mutant in System I cytochrome c biogenesis pathway
TMPD screen for cytochrome c defect in system II: in 1996, respiratory genomics told us that Bordetella would be a good model, leading to isolation of mutants in system II.
Evolution

System I produces more cytchrome c than system II when heme is limited

System I makes cytchromes c even when heme biosynthes is completely inhibited due to the heme reservoir on CcmE. We conclude that system I has advantages when environmental iron (therefore heme) is limiting.
Acknowledgements