Characterization of the Bradyrhizobium japonicum CycY Protein, a Membrane-anchored Periplasmic Thioredoxin That May Play a Role as a Reductant in the Biogenesis of c-Type Cytochromes

Abstract

A new member of membrane-anchored periplasmic thioredoxin-like proteins was identified in Bradyrhizobium japonicum. It is the product of cycY, the last gene in a cluster of cytochrome c biogenesis genes. Mutational analysis revealed that cycY is essential for the biosynthesis of all c-type cytochromes in this bacterium. The CycY protein was shown to be exported to the periplasm by its N-terminal signal sequence-like domain. Results from Western blot analyses of membrane and soluble fractions indicated that the CycY protein remains bound to the membrane. A soluble version of the protein devoid of its N-terminal membrane anchor (CycY*) was expressed in Escherichia coli and purified to homogeneity from the periplasmic fraction. The protein showed redox reactivity and properties similar to other thioredoxins such as fluorescence quenching in the oxidized form. Its equilibrium constant with glutathione was determined to be 168 mM, from which a standard redox potential of ؊0.217 V was calculated, suggesting that CycY might act as a reductant in the otherwise oxidative environment of the periplasm. This is in agreement with our hypothesis that CycY is required, directly or indirectly, for the reduction of the heme-binding site cysteines in the CXXCH motif of ctype apocytochromes before heme attachment occurs.

The Acidic Nature of the CcmG Redox-Active Center Is Important for CytochromecMaturation inEscherichia coli

“…However, the redox activity of CcmG is different from that of TRX and TRX-like proteins in that it is highly specific and limited to cytochrome c maturation (10,23). Furthermore, CcmG, unlike other TRX-like proteins, is not a catalyst of the insulin reduction assay (10). In addition, CcmG is reduced by the transmembrane electron transfer protein DsbD, supporting the notion that it plays a reducing role in cytochrome c maturation (14).…”

“…TRX maintains a reducing environment in the cytoplasm by reverting disulfide bonds to dithiols in cytoplasmic proteins (12). However, the redox activity of CcmG is different from that of TRX and TRX-like proteins in that it is highly specific and limited to cytochrome c maturation (10,23). Furthermore, CcmG, unlike other TRX-like proteins, is not a catalyst of the insulin reduction assay (10).…”

“…Probably the most convincing evidence is that CcmG homologues contain a conserved Cys-X-X-Cys motif that is redox active (10) and that is required for its role in cytochrome c maturation in vivo (8). The Cys-X-X-Cys motif is characteristic of thioredoxin-like (TRX-like) proteins, of which thioredoxin (TRX) is the archetype.…”

Gram-positive DsbE Proteins Function Differently from Gram-negative DsbE Homologs

“…In comparison with the standard redox potential for E. coli DsbA (Ϫ124 mV) (46), which is an oxidant, and E. coli thioredoxin (Ϫ269 mV) (35), which is a reductant, the standard redox potential for Mtb DsbE (Ϫ128 mV) suggests that DsbE is an oxidant. In contrast, the standard redox potentials for Gram-negative DsbE proteins (Ϫ217 to Ϫ175 mV) (47)(48)(49) correspond to these proteins being weak reductants. Since Mtb DsbE is an oxidant and Gram-negative DsbE proteins are weak reductants, this reinforces the hypothesis that Gram-negative and Gram-positive DsbE proteins function differently.…”

Bcl-2 prevents topoisomerase II inhibitor GL331-induced apoptosis is mediated by down-regulation of poly(ADP-ribose)polymerase activity

“…Recently, Bcl-2, which was found to locate on the outer membrane of mitochondria, not only prevents the initiation of the cellular apoptotic program by stabilizing the mitochondrial permeability transition (PT), but also avoids the subsequent release of cytochrome C and AIF protease from mitochondria (Susin et al, 1997). In addition to cytochrome C and AIF, PT pore also cause matrix Ca 2+ out¯ow (Sokolove and Haley, 1996), depletes the reduced glutathione (Fabianek et al, 1997), and alters the pyridine nucleotide pool (NADH 2 / NAD+NADPH 2 /NADP) (Takahashi et al, 1996). In this respect, Bcl-2 likely inhibits PARP activity through modulating PT pore and prevents mitochondrial NAD out¯ow into nucleus.…”