The Sco protein from Thermus thermophilus has previously been shown to perform a disulfide bond reduction in the Cu protein from T. thermophilus, which is a soluble protein engineered from subunit II of cytochrome ba oxidase that lacks the transmembrane helix. The native cysteines on TtSco and TtCu were mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine in TtCu and subsequent release upon incubation with the complementary TtSco protein demonstrated the formation of the mixed disulfide intermediate. The cysteine of TtSco that attacks the disulfide bond in the target TtCu protein was determined to be TtSco Cysteine 49. This cysteine is likely more reactive than Cysteine 53 due to a higher degree of solvent exposure. Removal of the metal binding histidine, His 139, does not change MDI formation. However, altering the arginine adjacent to the reactive cysteine in Sco (Arginine 48) does alter the formation of the MDI. Binding of Cu or Cu to TtSco prior to reaction with TtCu was found to preclude formation of the mixed disulfide intermediate. These results shed light on a mechanism of disulfide bond reduction by the TtSco protein and may point to a possible role of metal binding in regulating the activity. IMPORTANCE: The function of Sco is at the center of many studies. The disulfide bond reduction in Cu by Sco is investigated herein and the effect of metal ions on the ability to reduce and form a mixed disulfide intermediate are also probed.

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The Cu A center is the initial electron acceptor in cytochrome c oxidase, and it consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as they may influence the electronic and redox properties of the metal center. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the Cu A site (the TtCu A protein) was treated with the chemical modifier diethyl pyrocarbonate (DEPC) and the reaction followed through UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies at pH 5.0-9.0. A mutant protein (H40A/H117A) with the non-ligating histidines removed was similarly tested. Introduction of an electron-withdrawing DEPC-modification onto the ligating histidine 157 of TtCu A increased the reduction potential by over 70 mV, as assessed by cyclic voltammetry. Results from both proteins indicate that DEPC reacts with one of the two ligating histidines, modification of a ligating histidine raises the reduction potential of the Cu A site, and formation of the DEPC adduct is reversible at room temperature. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCu A through kinetically-controlled proton exchange with the solvent. Lack of reactivity by the metalloproteins Sco and azurin, both of which contain a mononuclear copper center, indicate that reactivity toward DEPC is not a characteristic of all ligating histidines.

Cytochrome c oxidase (CcO) is the final complex of the respiratory electron transport chain, which is a set of complexes that participate in the production of adenosine triphosphate (ATP), the main form of energy used by cells. CuA is the initial electron acceptor of CcO and consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine and the carbonyl backbone of a nearby glutamine. Sco proteins have been implicated in the assembly of the CuA, site but the exact role that it plays is the subject of much debate and study. Previous work has found that Sco proteins share a fold similar to thioredoxins, pointing toward the possibility of thiol‐disulfide oxidoreductase activity as a function of the protein. The ability of Thermus thermophilus Sco (TtSco) to act as a thiol‐disulfide oxidoreductase has been probed using an experiment to trap the protein‐protein intermediate between TtSco and its target, Thermus thermophilus CuA (TtCuA). The effect of nearby amino acids and metal binding prior to reaction have been determined for the formation of the protein‐protein intermediate, the mixed disulfide intermediate (MDI). The formation of MDI was monitored with either UV‐Visible spectroscopy by observing an absorbance increase at 412 nm as a chromophore tag, TNB, was released upon reaction between TtSco and TtCuA or by non‐reducing SDS‐PAGE gels. Altering Arg 48, which is adjacent to the attacking cysteine to an Asp has a very large effect on MDI production. TtSco also has metal binding ability, found to bind Cu(I), Cu(II), Ni(II), Cd(II) and Co(II) with varying relative affinities, in addition to disulfide reductase activity. To further characterize MDI formation with TtSco, the impact of conserved residues involved in metal binding on MDI formation were probed and showed that changing the conserved histidine did not affect MDI formation. However, binding of metal to Sco prior to incubating with TtCuA abrogated MDI formation. In characterizing the metal bound protein, preliminary studies to determine the binding affinity of Cu(II) to TtSco have been completed. The impact of conserved cysteines on metal binding has also been studied and the results indicate that one cysteine binds much more tightly to the Cu(II) than the other. While this work allows for a better understanding of TtSco function, much work remains to be done to understand the impacts of metal binding and to further characterize the Sco family of proteins.Support or Funding InformationResearch Corporation (Cottrell College Science Award 7963), the Welch Foundation (W‐0031 [Trinity Chemistry Department]), FASTER grant SURF‐ National Science Foundation DUE S‐STEM Award 1153796 and the Arnold and Mabel Beckman Foundation Beckman Scholars AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.