Heme-dependent regulation of bacterial cytochromes

Abstract

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 c 1 , a subunit of the cytochrome bc 1 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 c 550 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 c 550 polypeptide expression. However, apoproteins of cytochrome b 562 or Vitreoschilla hemoglobin accumulated independently of heme. Mutations within the conserved CXXCH heme-binding motif of cytochrome c 550 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 c 550 is degraded by the periplasmic protease DegP under heme limitation. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b 562 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.