| The heme prosthetic group of heme proteins contains iron, which can be a limiting nutrient. In the current study, we found that expression of B. japonicum cytochrome c1, a subunit of the cytochrome bc1 complex, is positively regulated by iron even though the mRNA steady state level is not controlled by iron availability. Genetic and biochemical evidence reveal that this iron-mediated control is mediated by the heme status. Since it has been well established that the ligation of heme to bacterial cytochrome c apoprotein occurs in periplasm, we hypothesize that heme-dependent regulation of cytochrome c protein is mediated by periplasmic protease(s), which degrade(s) cytochrome c apoprotein under heme limitation.; In order to address this question more easily, we took advantage of a simpler Escherichia coli system in which B. japonicum cytochrome c550 was expressed in the presence of a plasmid-borne class I cytochrome c maturation operon. We showed that heme deficiency leads to loss of B. japonicum c550 polypeptide expression. However, apoproteins of cytochrome b562 or Vitreoschilla hemoglobin accumulated independently of heme. Mutations within the conserved CXXCH heme-binding motif of cytochrome c550 or absence of Ccm also resulted in a low apoprotein accumulation. In each of these cases, protein levels were restored in a degP mutant strain, suggesting that apocytochrome c550 is degraded by the periplasmic protease DegP under heme limitation. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. Interestingly, this variant was stable in heme-deficient cells, but degraded in the absence of Ccm by DegP. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm-dependent nevertheless. We suggest that the cytochrome c heme binding motif is an instability element, and that stabilization by Ccm does not require ligation of the heme moiety to the protein. |
