Metal and redox selectivity of protoporphyrin binding to the heme chaperone CcmE

Abstract

The interaction of heme with the heme chaperone CcmE is central to our understanding of cytochrome c maturation, a complex post-translational process involving at least eight proteins in many Gram-negative bacteria and plant mitochondria. We have shown previously that Escherichia coli CcmE can interact with heme non-covalently in vitro, before forming a novel covalent histidine-heme bond, in a redox-sensitive manner. The function of CcmE is to bind heme in the periplasm before transferring it to apocytochromes c. In the absence of structural information on the complex of CcmE and heme, we have further characterized it by examining the binding of the soluble domain of CcmE (CcmE') to protoporphyrins containing metals other than Fe, namely Zn-, Sn-, Co- and Mn-protoporphyrin (PPIX). CcmE' demonstrated no affinity for the Zn- or Sn-containing protoporphyrins and low affinity for Mn(ii)-PPIX. High-affinity, reversible binding was, however, observed for Co(iii)-PPIX, which was highly sensitive to oxidation state as demonstrated by release of the ligand from the chaperone on reduction; no binding to Co(ii)-PPIX was observed. The non-covalent complex of CcmE' and Co(iii)-PPIX was characterized by non-denaturing mass spectrometry. The implications of these observations for the in vivo function of CcmE are discussed.

Molecular strategies of microbial iron assimilation: from high-affinity complexes to cofactor assembly systems

“…158,159 The membrane-attached chaperone forms a covalent heme adduct which results in cofactor oxidation, and transfers the ferric heme to the cytochrome c synthetase complex CcmF/H, which re-reduces the heme prior to its ligation to the target protein. 158,160 In addition to periplasmic heme chaperoning, an ankyrin-containing protein in Campylobacter is proposed to act as a heme chaperone for cytosolic heme trafficking and targeting during intracellular catalase maturation. 161 Relatively little is known about directed insertion mechanisms of iron into mononuclear sites such as in non-heme iron dioxygenases or into binuclear sites like diiron-oxo centers associated with ferroxidase activity.…”

Radical Mediated Rapid In Vitro Formation of c-Type Cytochrome

“…However, pathway B would be more favorable as it passes through a much more stable secondary radical intermediate adjacent to the porphyrin ring. This is contrary to the in vivo formation of Cyt c , which has a thioether bond produced through the thermodynamically unfavorable pathway A in the current reaction scheme [ 4 , 5 , 32 , 33 , 34 ]. This highlights the unusual selectivity of the enzymatic thioether formation in Cyt c , which is believed to require 10 accessory proteins (namely Cyt c maturation proteins) [ 4 , 5 , 34 ].…”

Binding of Immunoglobulin G to Protoporphyrin IX and Its Derivatives: Evidence the Fab Domain Recognizes the Protoporphyrin Ring

“…PPIX and Zn-PPIX were prepared using the same method described above for the preparation of hemin. Concentrations of hemin and Zn-PPIX were determined based on the molar extinction coefficient (ε 385 nm = 58,400 M −1 ·cm −1 and ε 412 nm = 87,400 M −1 ·cm −1 for hemin and Zn-PPIX, respectively), as previously described [11,12]. The PPIX concentration was also determined based on its molar extinction coefficient (ε 405 nm = 124,000 M −1 ·cm −1 ) after dissolving in dimethyl formamide [13].…”