H135A Controls the Redox Activity of the Sco Copper Center. Kinetic and Spectroscopic Studies of the His135Ala Variant of Bacillus subtilis Sco

Abstract

Sco-like proteins contain copper bound by two cysteines and a histidine residue. Although their function is still incompletely understood, there is a clear involvement with the assembly of cytochrome oxidases which contain the Cu A center in subunit 2, possibly mediating the transfer of copper into the Cu A binuclear site. We are investigating the reaction chemistry of BSco, the homologue from B. subtilis. Our studies have revealed that BSco behaves more like a redox protein than a metallochaperone. The essential H135 residue which coordinates copper, plays a role in stabilizing the Cu(II) rather than the Cu(I) form. When H135 is mutated to alanine, the oxidation rate of both hydrogen peroxide and one-electron outer-sphere reductants increases by two orders of magnitude, suggestive of a redox switch mechanism between His-on and His-off conformational states of the protein. Imidazole binds to the H135A protein restoring the N superhyperfine coupling in the EPR, but is unable to rescue the redox properties of the WT Sco. These findings reveal a unique role for H135 in Sco function. We propose a hypothesis that electron transfer from Sco to the maturing oxidase may be essential for proper maturation and/or protection from oxidative damage during the assembly process. The findings also suggest that interaction of Sco with its protein partner(s) may perturb the Cu(II)-H135 interaction, and thus induce a sensitive redox activity to the protein.Sco1 is an essential accessory protein in the assembly of cytochrome-c-oxidase, the terminal enzyme of the respiratory chain. A large body of evidence links the function of Sco to the metalation of the Cu A center in subunit 2 of the oxidase (Cox2). In both yeast and B. subtilis, Sco mutant strains that impair or eliminate Cu binding produce a phenotype lacking in functional caa 3 oxidase, and high levels of Cu are able to rescue the caa 3 activity of B. subtilis-Sco (BSco) 1 -deficient strains (1-6). These data strongly implicate an interaction of † This work was supported in whole or in part by the National Institutes of Health Grant GM54803 (to NJB). This work was also supported 1 The abbreviations used are: BSco, Bacillus subtilis Sco; IPTG, isopropyl β-D-thiogalactopyranoside; WT, wild-type; NBT, nitroblue tetrazolium; BCIP, 5-bromo-4-chloro-3-indolylphosphate; TMPD, N, N, N′, N′-tetramethyl-p-phenylene diamine; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; EXAFS, extended X-ray absorption fine structure; XANES, X-ray absorption near edge structure. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 29. (14), and the Cu(II) form shows no tendency towards autoredox to disulfide and Cu(I), although this chemistry was observed in a crystal of the Ni(II) derivative (11). These considerations suggest that Sco-type proteins may exhibit two or more distinct activities which include both copper transfer and redox activities.To further understand the possible function of Sco ...

Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of Cu A in human cytochrome oxidase

“…Both proteins share a thioredoxin fold and bind copper ions through a functional CX 3 C motif complemented by a histidine residue located in a β-hairpin absent in thioredoxins (24)(25)(26)(27). This CX 3 CX n H motif allows Sco proteins either to bind Cu(I) with high affinity or to act as thioloxidoreductases (27)(28)(29). Both Sco1 and Sco2 are essential for Cu A biogenesis in humans, but their specific roles are unknown, and a detailed mechanism for Cu A assembly in mammalian oxidases is still lacking.…”

An Unconventional Copper Protein Required for Cytochrome c Oxidase Respiratory Function under Extreme Acidic Conditions

“…Another protein conserved among most of the living organisms and well known as essential for the CcO system is Sco1. The notion of a multifunctional protein is suggested for Sco-type proteins, which may exhibit two or more distinct roles (37,38). Sco1 has first been described as a metallochaperone involved in copper transfer to the CcO system.…”

“…Sco1 has first been described as a metallochaperone involved in copper transfer to the CcO system. More recently, it has been suggested that Sco1 might also behave like a redox protein (37); and a tight association with the fully assembled CcO complex suggests another unknown role than the transient post-translational CcO maturation protein (38). Even if there is no sequence similarity between Sco1 and AcoP, similar multifunctional properties are now demonstrated for both proteins.…”

Binuclear CuA Formation in Biosynthetic Models of CuA in Azurin Proceeds via a Novel Cu(Cys)2His Mononuclear Copper Intermediate

“…45–50 Quite interestingly, binding of both Cu(II) and Cu(I) to Sco1 has been proposed to be important for normal functioning of this protein. 51–53 Alternatively, it has been proposed that Sco1 acts as disulfide reductase to keep the active site Cys of Cu A in the reduced state or to maintain a proper redox buffering of metal ions. The latter inference stems from the fact that Sco1 possesses the thioredoxin fold.…”