Thioredoxin‐like protein TlpA from Bradyrhizobium japonicum is a reductant for the copper metallochaperone ScoI

Abstract

a b s t r a c tTlpA and ScoI of Bradyrhizobium japonicum are membrane-anchored thioredoxin-like proteins oriented towards the periplasm. TlpA is a protein-disulfide reductase. ScoI is a copper chaperone for cytochrome oxidase biogenesis. TlpA with its negative redox potential (E o 0 À256 mV) was shown here to reduce oxidized ScoI, for which we determined a less negative

How Periplasmic Thioredoxin TlpA Reduces Bacterial Copper Chaperone ScoI and Cytochrome Oxidase Subunit II (CoxB) Prior to Metallation*

“…Detailed information on the individual plasmids including the coding nucleotide and the corresponding protein sequences is available from the authors on request. For production of ScoI S C74S , the previously described plasmid pRJ8336 (20) was used.…”

“…TlpA S , used for the disulfide exchange equilibrium with CoxB PD , and TlpA S C110S , used for crystallization, were produced as fusions to the C terminus of maltose-binding protein (MalE) and purified after factor Xa cleavage from MalE as described previously (16,20,23). Alternatively, an analogous expression plasmid (pMal-p/TEV-TlpA S ) encoding the same fusion with a TEV protease instead of a factor Xa cleavage site was used for production of TlpA S for stopped-flow kinetics.…”

“…TlpA S purified with this alternative method bears two additional glycines at its N terminus, which proved to be necessary for efficient cleavage (calculated mass: 19,634.7 Da; found: 19,632.5 Da). ScoI S C74S was produced and purified as reported recently (20). TEV protease was purified as described (24).…”

“…Eluted proteins were detected at 220 nm, and their concentration was quantified by peak integration. The equilibrium constant (K eq ) and the redox potential of CoxB PD were calculated according to Equation 1 and the Nernst equation (Equation 2), respectively, using a value of Ϫ256 mV for the redox potential (EЈ 0 ) of TlpA S (20).…”

“…The Cu 2ϩ -binding domain of the membrane-anchored ScoI and Sco1 proteins faces the periplasm and intermembrane space, respectively, akin to CoxB. We had shown recently that TlpA serves as a reductant for ScoI (20). With few exceptions, the biogenesis of the Cu A center in eukaryotic mitochondria and in prokaryotes depends on the presence of Sco1/ScoI (18,19).…”