Brian San Francisco

ENVIRONMENTAL IRON LIMITATION: BENEFITS TO AN ORGANISM WITH A SYSTEM I VERSUS A SYSTEM II CYTOCHROME C BIOGENESIS PATHWAY. Brian San Francisco¹, Robert Kranz¹, Biology Department, Washington University, St. Louis, MO.¹

Cytochromes are electron transport proteins found in organisms from every kingdom of life. c-type cytochromes are distinguished from other cytochromes by the covalent attachment of a heme moiety (central iron atom bound to a protoporphyrin ring) to specific cysteine residues in the heme-binding site (Cys-X-X-Cys-His) of the apocytochrome. The attachment of the heme to the apoctyochrome occurs at the site of action (in bacteria, outside the cytoplasmic membrane), and thus presents unique challenges to synthesis. Three distinct systems (I, II, III) have evolved for cytochrome c biogenesis.

Systems I and II are found in bacteria, as well as some eukaryotes. These two pathways vary greatly in terms of complexity, but both accomplish 1) the reduction of specific cysteine residues of the apocytochrome c and the iron of heme, 2) the movement of the heme from its site of synthesis (the cytoplasm) across the cytoplasmic membrane, and 3) the ligation of the heme group to the apocytochrome c outside the cytoplasmic membrane. System II consists of four proteins, two of which (CcsA and CcsB) function specifically in heme transport and ligation to the apocytochrome. System I uses eight proteins (CcmA-H) including CcmE, a protein that functions as a heme chaperone capable of heme storage, and the CcmABCD, an ABC transporter-like release complex. The high affinity association of holo (heme) CcmE with CcmC requires ATP hydrolysis for release of the CcmE chaperone to the cytochrome c synthetase (CcmF/H).

All bacteria utilize either system I or system II. However, despite the widespread distribution of systems I and II, the environmental pressure that led to the evolution of these two distinct biogenesis pathways has yet to be identified. In order to compare both systems in a single genetic background, recombinant system I and system II plasmids were constructed. A strain of Escherichia coli was deleted for all eight system I genes involved in cytochrome c synthesis (∆ccm), and serves as the host for the system I plasmid (ccmABCDEFGH from E. coli) or the system II plasmid (fused ccsB/A gene from Helicobacter). Each recombinant system is under the control of an IPTG-inducible promoter. An arabinose-inducible cytochrome c₄ from Bordetella pertussis with a 6X Histidine tag (cyt c₄:His) allows for easy detection of cytochrome c biogenesis. Cytochrome c biogenesis was restored when the reporter plasmid and either system were induced in E. coli Dccm.

Using the recombinant cytochrome c biogenesis systems, it has been demonstrated that system I (via CcmABCD and CcmE) can synthesize c-type cytochromes at significantly lower heme levels than those required for system II cytochrome c biogenesis. Heme biosynthesis is the insertion of an iron atom into a protoporphyrin ring, and so the amount of heme available for cytochrome c biogenesis depends directly on the amount of available iron in the environment. We demonstrate here, using the iron chelator 2,2’ dipyridyl, that system I can synthesize c-type cytochromes at lower iron concentrations than system II. We suggest that the amount of environmentally available iron is a selective pressure that allowed for the evolution of the system I and system II cytochrome c biogenesis pathways.