Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia.

BACKGROUND: It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met(80) to Met(80) sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met(80)-Fe ligation of cyt c, forming pentacoordinated heme Fe(2+), which inhibits electron transport (ET) and promotes oxygenase activity. METHODS: SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met(80)-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy. RESULTS: Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met(80)-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20. CONCLUSION: Our results show that ischemia disrupts the Met(80)-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe(2+) that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release. GENERAL SIGNIFICANCE: Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met(80)-Fe ligation, minimizes ischemic injury and promotes ATP recovery.

Transport of sucrose, not hexose, in the phloem.

Several lines of evidence indicate that glucose and fructose are essentially absent in mobile phloem sap. However, this paradigm has been called into question, especially but not entirely, with respect to species in the Ranunculaceae and Papaveraceae. In the experiments in question, phloem sap was obtained by detaching leaves and placing the cut ends of the petioles in an EDTA solution. More hexose than sucrose was detected. In the present study, these results were confirmed for four species. However, almost identical results were obtained when the leaf blades were removed and only petiole stubs were immersed. This suggests that the sugars in the EDTA solution represent compounds extracted from the petioles, rather than sugars in transit in the phloem. In further experiments, the leaf blades were exposed to (14)CO(2) and, following a chase period, radiolabelled sugars in the petioles and EDTA exudate were identified. Almost all the radiolabel was in the form of [(14)C]sucrose, with little radiolabelled hexose. The data support the long-held contention that sucrose is a ubiquitous transport sugar, but hexoses are essentially absent in the phloem stream.