Mitochondria are central to energy metabolism as the source of much of the cell’s ATP, as well as being a hub for cellular Ca2+ signaling. Mitochondrial Ca2+ is a positive effector of ATP synthesis, yet Ca2+ overload can lead to mitochondrial dysfunction and cell death. Moreover, Ca2+ uptake by mitochondria is involved in shaping cellular Ca2+ dynamics by regulating the concentrations of Ca2+ within microdomains between mitochondria and sarco/endoplasmic reticulum and plasma membrane Ca2+ transporters. Reactive oxygen species (ROS) generated as a consequence of ATP production in the mitochondria are important for cellular signaling, yet contribute to oxidative stress and cellular damage. ROS regulate the activity of redox sensitive enzymes and ion channels within the cell, including Ca2+ channels. For both Ca2+ and ROS, a delicate balance exists between the beneficial and detrimental effects on mitochondria. In this review we bring together current data on mitochondrial Ca2+ uptake, ROS generation, and redox modulation of Ca2+ transport proteins. We present a model for crosstalk between Ca2+ and ROS signaling pathways within mitochondrial microdomains.

SummaryGenetic 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 c 4 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 (holoCcmE), 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.

SummaryAlthough 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 CcmA 2B1C1 complex and ATPdependent release of holoCcmE. We propose that the CcmA2B1C1 complex represents a new subgroup within the ABC transporter superfamily that functions to release a chaperone.

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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 (⌬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).