Elaine R. Frawley

Cytochrome c Biogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

SUMMARY Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe+3 state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe+2) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments.

Recombinant cytochromes c biogenesis systems I and II and analysis of haem delivery pathways in Escherichia coli.

Genetic analysis has indicated that the system II pathway for c‐type cytochrome biogenesis in Bordetella pertussis requires at least four biogenesis proteins (CcsB, CcsA, DsbD and CcsX). In this study, the eight genes (ccmA–H) associated with the system I pathway in Escherichia coli were deleted. Using B. pertussis cytochrome c4 as a reporter for cytochromes c assembly, it is demonstrated that a single fused ccsBA polypeptide can replace the function of the eight system I genes in E. coli. Thus, the CcsB and CcsA membrane complex of system II is likely to possess the haem delivery and periplasmic cytochrome c‐haem ligation functions. Using recombinant system II and system I, both under control of IPTG, we have begun to study the capabilities and characteristics of each system in the same organism (E. coli). The ferrochelatase inhibitor N‐methylprotoporphyrin was used to modulate haem levels in vivo and it is shown that system I can use endogenous haem at much lower levels than system II. Additionally, while system I encodes a covalently bound haem chaperone (holo‐CcmE), no covalent intermediate has been found in system II. It is shown that this allows system I to use holo‐CcmE as a haem reservoir, a capability system II does not possess.

ABC transporter-mediated release of a haem chaperone allows cytochrome c biogenesis

Although organisms from all kingdoms have either the system I or II cytochrome c biogenesis pathway, it has remained a mystery as to why these two distinct pathways have developed. We have previously shown evidence that the system I pathway has a higher affinity for haem than system II for cytochrome c biogenesis. Here, we show the mechanism by which the system I pathway can utilize haem at low levels. The mechanism involves an ATP‐binding cassette (ABC) transporter that is required for release of the periplasmic haem chaperone CcmE to the last step of cytochrome c assembly. This ABC transporter is composed of the ABC subunit CcmA, and two membrane proteins, CcmB and CcmC. In the absence of CcmA or CcmB, holo(haem)CcmE binds to CcmC in a stable dead‐end complex, indicating high affinity binding of haem to CcmC. Expression of CcmA and CcmB facilitates formation of the CcmA2B1C1 complex and ATP‐dependent release of holoCcmE. We propose that the CcmA2B1C1 complex represents a new subgroup within the ABC transporter superfamily that functions to release a chaperone.

CcsBA is a cytochrome c synthetase that also functions in heme transport

Little is known about trafficking of heme from its sites of synthesis to sites of heme-protein assembly. We describe an integral membrane protein that allows trapping of endogenous heme to elucidate trafficking mechanisms. We show that CcsBA, a representative of a superfamily of integral membrane proteins involved in cytochrome c biosynthesis, exports and protects heme from oxidation. CcsBA has 10 transmembrane domains (TMDs) and reconstitutes cytochrome c synthesis in the Escherichia coli periplasm; thus, CcsBA is a cytochrome c synthetase. Purified CcsBA contains heme in an “external heme binding domain” for which two external histidines are shown to serve as axial ligands that protect the heme iron from oxidation. This is likely the active site of the synthetase. Furthermore, two conserved histidines in TMDs are required for heme to travel to the external heme binding domain. Remarkably, the function of CcsBA with mutations in these TMD histidines is corrected by exogenous imidazole, a result analogous to correction of heme binding by myoglobin when its proximal histidine is mutated. These data suggest that CcsBA has a heme binding site within the bilayer and that CcsBA is a heme channel.

Iron and citrate export by a major facilitator superfamily pump regulates metabolism and stress resistance in Salmonella Typhimurium

The efficacy of antibiotics and host defenses has been linked to the metabolic and redox states of bacteria. In this study we report that a stress-induced export pump belonging to the major facilitator superfamily effluxes citrate and iron from the enteric pathogen Salmonella Typhimurium to arrest growth and ameliorate the effects of antibiotics, hydrogen peroxide, and nitric oxide. The transporter, formerly known as MdtD, is now designated IceT (iron citrate efflux transporter). Iron efflux via an iron-chelating tricarboxylic acid cycle intermediate provides a direct link between aerobic metabolism and bacterial stress responses, representing a unique mechanism of resistance to host defenses and antimicrobial agents of diverse classes.

The NsrR regulon in nitrosative stress resistance of Salmonella enterica serovar Typhimurium

Nitric oxide (NO·) is an important mediator of innate immunity. The facultative intracellular pathogen Salmonella has evolved mechanisms to detoxify and evade the antimicrobial actions of host‐derived NO· produced during infection. Expression of the NO·‐detoxifying flavohaemoglobin Hmp is controlled by the NO·‐sensing transcriptional repressor NsrR and is required for Salmonella virulence. In this study we show that NsrR responds to very low NO· concentrations, suggesting that it plays a primary role in the nitrosative stress response. Additionally, we have defined the NsrR regulon in Salmonella enterica sv. Typhimurium 14028s using transcriptional microarray, qRT‐PCR and in silico methods. A novel NsrR‐regulated gene designated STM1808 has been identified, along with hmp, hcp‐hcr, yeaR‐yoaG, ygbA and ytfE. STM1808 and ygbA are important for S. Typhimurium growth during nitrosative stress, and the hcp‐hcr locus plays a supportive role in NO· detoxification. ICP‐MS analysis of purified STM1808 suggests that it is a zinc metalloprotein, with histidine residues H32 and H82 required for NO· resistance and zinc binding. Moreover, STM1808 and ytfE promote Salmonella growth during systemic infection of mice. Collectively, these findings demonstrate that NsrR‐regulated genes in addition to hmp are important for NO· detoxification, nitrosative stress resistance and Salmonella virulence.

Heme Concentration Dependence and Metalloporphyrin Inhibition of the System I and II Cytochrome c Assembly Pathways

Studies have indicated that specific heme delivery to apocytochrome c is a critical feature of the cytochrome c biogenesis pathways called system I and II. To determine directly the heme requirements of each system, including whether other metal porphyrins can be incorporated into cytochromes c, we engineered Escherichia coli so that the natural system I (ccmABCDEFGH) was deleted and exogenous porphyrins were the sole source of porphyrins (Delta hemA). The engineered E. coli strains that produced recombinant system I (from E. coli) or system II (from Helicobacter) facilitated studies of the heme concentration dependence of each system. Using this exogenous porphyrin approach, it was shown that in system I the levels of heme used are at least fivefold lower than the levels used in system II, providing an important advantage for system I. Neither system could assemble holocytochromes c with other metal porphyrins, suggesting that the attachment mechanism is specific for Fe protoporphyrin. Surprisingly, Zn and Sn protoporphyrins are potent inhibitors of the pathways, and exogenous heme competes with this inhibition. We propose that the targets are the heme binding proteins in the pathways (CcmC, CcmE, and CcmF for system I and CcsA for system II).

Bacterial Stress Responses during Host Infection

Pathogenic bacteria must withstand diverse host environments during infection. Environmental signals, such as pH, temperature, nutrient limitation, etc., not only trigger adaptive responses within bacteria to these specific stress conditions but also direct the expression of virulence genes at an appropriate time and place. An appreciation of stress responses and their regulation is therefore essential for an understanding of bacterial pathogenesis. This review considers specific stresses in the host environment and their relevance to pathogenesis, with a particular focus on the enteric pathogen Salmonella.

The ins and outs of bacterial iron metabolism.

Iron is a critical nutrient for the growth and survival of most bacterial species. Accordingly, much attention has been paid to the mechanisms by which host organisms sequester iron from invading bacteria and how bacteria acquire iron from their environment. However, under oxidative stress conditions such as those encountered within phagocytic cells during the host immune response, iron is released from proteins and can act as a catalyst for Fenton chemistry to produce cytotoxic reactive oxygen species. The transitory efflux of free intracellular iron may be beneficial to bacteria under such conditions. The recent discovery of putative iron efflux transporters in Salmonella enterica serovar Typhimurium is discussed in the context of cellular iron homeostasis.

Topology and Function of CcmD in Cytochrome c Maturation

The system I cytochrome c biogenesis pathway requires CcmD, a small polypeptide of 69 residues in Escherichia coli. Here it is shown that CcmD is a component of the CcmABC ATP-binding cassette transporter complex. CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE in the periplasm or for interaction of CcmE with CcmABC. CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex. Evidence is presented that the topology of CcmD in the cytoplasmic membrane is the N terminus outside and the C terminus inside with one transmembrane domain.