Characterization and Effect of Metal Ions on the Formation of the \u3cem\u3eThermus thermophilus\u3c/em\u3e Sco Mixed Disulfide Intermediate

The Sco protein from Thermus thermophilus has previously been shown to perform a disulfide bond reduction in the CuA protein from T. thermophilus, which is a soluble protein engineered from subunit II of cytochrome ba 3 oxidase that lacks the transmembrane helix. The native cysteines on TtSco and TtCuA were mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine in TtCuA 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 TtCuA 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 Cu2+ or Cu+ to TtSco prior to reaction with TtCuA 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 CuA 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

Reactive Sites and Course of Reduction in the Rieske Protein

Rieske proteins play an essential role in electron transfer in the bc1 complex. Rieske proteins contain a [2Fe–2S] cluster with one iron ligated by two histidines and the other iron ligated by two cysteines. All Rieske proteins have pH-dependent reduction potentials with the histidines ligating the cluster deprotonating in response to increases in pH. The addition of diethylpyrocarbonate (DEPC) modifies deprotonated histidines. The previous studies on the isolated Thermus thermophilus Rieske protein have used large excesses of DEPC, and this study examines what amino acids become modified under different molar equivalents of DEPC to protein. Increasing amounts of DEPC result in more modification, and higher pH values result in faster reaction. Upon modification, the protein also becomes reduced and ~6 equivalents of DEPC are needed for 50% of the reduction to occur. Which amino acids are modified first also points to the most reactive species on the protein. Mass spectrometry analysis shows that lysine 68 is the most reactive amino acid, followed by the ligating histidine 154 and two other surfaces lysines, 76 and 43. The modification of the ligating histidine at low numbers of DEPC equivalents and correlation with a similar number of equivalents needed to reduce the protein shows that this histidine can interact with neighboring groups, and these results can be extended to the protein within the bc1 complex, where interaction with neighboring residues or molecules may allow reduction to occur. These results may shed light on how Rieske transfers electrons and protons in the bc1 complex

DEPC modification of the CuA protein from Thermus thermophilus

The CuA 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 CuA site (the TtCuA 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 TtCuA 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 CuA 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 TtCuA 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

Spectroscopic Characterization of Mn\u3csup\u3e2+\u3c/sup\u3e and Cd\u3csup\u3e2+\u3c/sup\u3e Coordination to Phosphorothioates in the Conserved A9 Metal Site of the Hammerhead Ribozyme

Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-SRp substitution is inhibitory in Mg2+, thiophilic Cd2+ rescues HHRz activity. Mn2+ is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-SRp HHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn2+ and Cd2+ coordination to Rp and Sp phosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd2+ does indeed bind to S in the thio-substituted ligand, Mn2+ coordinates to the non‑sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of Mn–O over Mn–S coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn2+ coordination preference of oxygen in either Rp or Sp A9 phosphorothioates differentially tunes catalytic activity, with Mn–O coordination in the A9-SRp phosphorothioate enzyme being inhibitory