Probing a Complex of Cytochromecand Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH–-ligated. The ferrous state is predominantly high-spin and, most likely, His/–. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form

Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism

AbstractDuodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b561 family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at gmax=3.7 corresponding to a highly anisotropic species, and another at gmax=3.18; these species are similar to those observed in other cytochromes of the b561 family, and were reducible by ascorbate. In addition another signal was observed in some preparations at gmax=2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of +80 mV±30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b561 of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes

Macroporous materials as novel catalysts for efficient and controllable proteolysis

A novel nanopore based digestion strategy has been developed by directly adding a macroporous material as catalyst to the conventional in-solution reaction system. Without increasing the enzyme or protein concentrations, this simple digestion approach exhibits high proteolysis efficiency and selectivity due to the in situ fast adsorption of both enzymes and proteins from bulk solution into the macropores of the catalysts, where the target substrates and enzymes are greatly concentrated and confined in the nanospace to realize a quick digestion. Based on the electrostatic interaction matching between the biomolecules and catalysts, selective extraction and digestion of proteins with different isoelectric points can be achieved by adjusting the surface charge of the catalysts. This nanoporous reaction system has been successfully applied to the analysis of a complex biological sample, where 293 proteins are identified, while only 100 proteins are obtained by the standard overnight in-solution digestion. The present nanospace confined digestion strategy will lead to promising advances not only in proteomics but also in other applications where enzymatic reactions are involved