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

A new member of membrane-anchored periplasmic thioredoxin-like protein s was identified in Bradyrhizobium japonicum. It is the product of cyc Y, the last gene in a cluster of cytochrome c biogenesis genes. Mutati onal analysis revealed that cycY is essential for the biosynthesis of all c-type cytochromes in this bacterium. The CycY protein was shown t o be exported to the periplasm by its N-terminal signal sequence-like domain. Results from Western blot analyses of membrane and soluble fra ctions indicated that the CycY protein remains bound to the membrane. A soluble version of the protein devoid of its N-terminal membrane anc hor (CycY) was expressed in Escherichia coli and purified to homogene ity from the periplasmic fraction. The protein showed redox reactivity and properties similar to other thioredoxins such as fluorescence que nching 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 reductan t in the otherwise oxidative environment of the periplasm. This is in agreement with our hypothesis that CycY is required, directly or indir ectly, for the reduction of the heme-binding site cysteines in the CXX CH motif of c-type apocytochromes before heme attachment occurs.