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 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 c-type apocytochromes before heme attachment occurs.
Fabianek
R A
RA
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland.
Huber-Wunderlich
M
M
Glockshuber
R
R
Künzler
P
P
Hennecke
H
H
Thöny-Meyer
L
L
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
D003574:Cytochrome c Group; D011994:Recombinant Proteins; C080693:cytochrome Cy; D013879:Thioredoxins