The mitochondrial copper chaperone COX11 plays an auxiliary role in the defence against oxidative stress

COX11, a protein anchored in the inner mitochondrial membrane, was originally identified as a copper chaperone delivering Cu+ to the cytochrome c oxidase of the respiratory chain. Here, we present evidence that this protein is also involved in the defence against reactive oxygen species. Quantitative PCR analyses in the model plant Arabidopsis thaliana revealed that the level of AtCOX11 mRNA rises under oxidative stress. The unexpected result that AtCOX11 knock-down lines contained less ROS than the wild-type can possibly be explained by the impaired oxidative phosphorylation, resulting in less respiration-dependent ROS formation. Similarly, we observed that yeast Saccharomyces cerevisiae ScCOX11 null mutants produced less ROS than wild-type cells. However, when exposed to oxidative stress, yeast strains overexpressing ScCOX11 or AtCOX11 showed lower ROS levels compared with the control indicating a ROS-detoxifying effect of the COX11 proteins. The additive effect on ROS sensitivity upon deletion of ScCOX11 in addition to the known ROS scavenger gene SOD1 encoding superoxide dismutase 1 corroborates the oxidative stress-relieving function of ScCOX11. Moreover, yeast strains overexpressing soluble versions of either AtCOX11 or ScCOX11 became more resistant against oxidative stress. The importance of three conserved cysteines for the ROS scavenger function became apparent after their deletion that resulted in the loss of ROS resistance. Further studies of strains producing COX11 proteins with individually mutated cysteines indicate that the formation of disulphide bridges might be the underlying mechanism responsible for the antioxidative activity of COX11 proteins. Both AtCOX11 and ScCOX11 apparently partake in oxidative stress defence by directly or indirectly exploiting the redox capacity of their cysteine residues.


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For many organisms, aerobic cellular respiration is an essential process, which 50 converts chemical energy stored in sugars and other metabolites into ATP. This complex 51 process is completed by the mitochondrial electron transport chain which shuttles 52 electrons from NAD(P)H and succinate to the terminal acceptor, molecular oxygen [1]. 53 During this process some electrons escape and reduce molecular oxygen, generating 54 superoxide, which can subsequently be converted into other reactive oxygen species 55 (ROS) [2]. While respiratory complexes represent a major source of ROS in mitochondria, 56 several other redox reactions also contribute to ROS production [3]. It is estimated that 57 1-5% of molecular oxygen is converted to ROS [4]. 58 ROS molecules are highly reactive and can oxidize and thereby damage other 59 molecules such as lipids, proteins, and nucleic acids. Consequently, organisms have 60 evolved complex mechanisms to control ROS levels and reduce their toxicity and 61 detrimental effects (reviewed in [2] and [3]). Some of them are well characterised, for 62 example, the enzyme family of superoxide dismutases (SOD), which convert superoxide 63 ions into oxygen and hydrogen peroxide [5]. The contribution of other proteins to oxidative 64 defence is less well understood and often speculative. One such example is the COX11 However, members of this conserved protein family might be directly involved in 81 mitochondrial oxidative metabolism, as suggested by several publications [13,14,15,16]. 82 Pungartnik et al. [13] showed that the yeast Sccox11 null mutant is highly sensitive to the 83 ROS inducing chemicals N-nitrosodiethylamine and 8-hydroxyquinoline. Subsequently, 84 Khalimonchuk et al. [14] and Veniamin et al. [15] demonstrated that the ΔSccox11 strain 85 also showed an increased sensitivity to hydrogen peroxide when compared with the WT 86 strain. For the rice (Oryza sativa) COX11 homologue (OsCOX11), direct scavenging of 87 ROS was suggested [16]. The authors reported that OsCOX11 dysfunction leads to a 88 loss of pollen viability, presumably because the timing of a ROS burst necessary for pollen 89 maturation is disturbed. Our previous investigation on AtCOX11 also hinted at its 90 contribution to ROS homeostasis during pollen germination [11]: both the AtCOX11 KD 91 and OE lines exhibited reduced pollen germination rates, which did not correlate with the 5 92 observed changes in COX activity, suggesting that AtCOX11 may have an additional 93 function during pollen germination besides COX assembly. 94 However, the role of COX11 in ROS homeostasis remained elusive. Here, we present 95 our data of a more detailed investigation of COX11's involvement in oxidative metabolism. 96 Our results indicate that both Arabidopsis and yeast COX11 partake in oxidative stress 97 defence, possibly directly by scavenging ROS.  YPL132w::kanMX4) was generated by crossing the respective single-deletion strains 118 followed by sporulation, tetrad dissection and analysis.

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Constructs used for COX11 overexpression (pAG415ADH-AtCOX11 and 120 pAG415ADH-ScCOX11) were generated previously [11]. To create the soluble versions 121 of COX11, fragments were amplified by PCR (for primer sequences and cloning details 122 see S1 Table) and inserted by Gateway cloning into pDONR or pENTR vectors. All 123 constructs were moved into the high-copy yeast expression-vector pAG425GPD-ccdB-124 EGFP [17]. Yeast cells were transformed as described in Gietz Table. 11 226 227 The overrepresentation of putative ROS-responsive elements prompted us to 228 analyse the expression levels of AtCOX11 transcripts under oxidative stress ( Fig 1B). We 229 treated WT seedlings for 2 h or 6 h with the oxidative reagents hydrogen peroxide (H 2 O 2 ), 230 tert-butyl hydroperoxide (t-BOOH) and antimycin A followed by qPCR analyses.   1C). On the other hand, its homologue AtHCC2 248 (homologue of copper chaperone SCO2), which lacks a copper-binding motif [38], was affected by ROS. It showed a decrease of the transcript level by about half (Fig 1C), even 250 though its promoter region carries seven putative ROS-responsive elements (S1 Fig).

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AtHCC2 levels, 2.2 times higher after a 6-h antimycin A treatment, were a notable 252 exception from the otherwise observed downregulation. Although the AtHCC2 expression 253 pattern was different from AtCOX11, the fact that AtHCC2 responded to ROS fits a 254 previously proposed role of AtHCC2 in redox homeostasis [38,39]. 255 The transcript levels of another COX-related gene, the COX subunit AtCOX5b-1, 256 were reduced by ~30% after 2 h of oxidative stress and by ~50% after 6 h, except for the 257 H 2 O 2 treatment, which had no effect at this time point (Fig 1C). Clearly, not all  Table).  Table. 290 291 MDA and HAE levels were lower in all KD lines compared with the WT, albeit only 292 statistically significant for KD1-1 and KD1-2 plants (Fig 2A). The levels in the OE lines 293 were indistinguishable from the WT.
14 294 These data were confirmed by a second assay, in which protoplasts were incubated 295 with the DCFDA dye, which upon entering the cell and oxidation by ROS exhibits a bright 296 green fluorescence. All KD lines showed a statistically significant reduction in cellular 297 ROS levels compared with the WT and again, the OE lines were indistinguishable from 298 the WT (Fig 2B). Of note is that these assays detect ROS from the entire cell and might 299 not be sensitive enough to detect subtle changes in the intermembrane space of 300 mitochondria.

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These results seemingly contradict a function of Arabidopsis COX11 in ROS defence.  Like in plants (Fig 2), the ScCOX11 knock-out (KO; ΔSccox11) strain showed a 335 significant reduction in the cellular ROS levels compared with the WT strain ( Fig 3A). The 336 overexpression of either the yeast or plant COX11 protein did not affect ROS levels 337 compared with the control strain transformed with the empty vector (Fig 3B) (Fig 3A). The same treatment did not affect ROS levels in the respiratory deficient 343 ScCOX11 KO strain (Fig 3A). The AtCOX11 or ScCOX11 overexpressing yeast strains 344 showed increased ROS levels in response to PQ (Fig 3B). However, the ROS levels' 345 increase was slightly, but significantly smaller compared with the increase in the empty-346 vector control (Fig 3B). This indicates that the overexpression of COX11 genes can partly    these constructs and their growth monitored on YPD plates (Fig 4B). Menadione was 396 chosen as the oxidative stressor because it is a known general redox cycler and ROS 397 inducer in the cytoplasm and other compartments [44].

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All yeast strains grew equally well in the absence of oxidative stress. When 399 menadione was added to the medium, however, the empty-vector, as well as the GFP-400 expressing controls, were almost unable to maintain growth, even at the lowest 401 menadione concentration (Fig 4B). The halted growth of the GFP control shows that the 402 overexpression of a random protein does not confer oxidative stress tolerance.

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The yeast strains expressing either AtCOX11 sol or ScCOX11 sol , however, continued 404 to grow at all three menadione concentrations tested (Fig 4B). At the lowest concentration 405 of 110 µM, growth remained almost unaffected. These results indicate that the increased 19 406 menadione tolerance in yeast expressing soluble COX11 is likely linked to some intrinsic 407 feature(s) of the COX11 proteins.

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What is the feature that allows COX11 proteins to heighten resistance to oxidative 409 stress? One possibility would be the three highly conserved cysteines present in COX11 410 proteins, of which two belong to the copper-binding motif (Fig 4A and S3 Fig) [7]. There

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To find out which of the three possible bridges (labelled "a", "b" and "c" in the 422 schematic illustrations in Fig 4B) might be involved, we generated six more constructs, 423 three Arabidopsis and three yeast COX11 versions, in which in each case one of the three 424 cysteines was mutated to an alanine thus restricting the number of putative disulphide 425 bridges that can be formed (illustrated in the schemes on the right of Fig 4B). The yeast The role of COX11 proteins as copper chaperones in COX complex assembly has 440 been well documented [8,9,11,12,45]. In this work, we present evidence that COX11 441 proteins have an auxiliary role in the defence against oxidative stress.

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The initial hint for such a role came from our observation that the expression of the 443 Arabidopsis COX11 gene was upregulated in response to oxidative stress ( Fig 1B). This 444 appeared to be a specific response of the AtCOX11 gene and not part of a general 445 upregulation of mitochondrial genes because AtHCC1 levels, for example, remained 446 unchanged and AtHCC2 and AtCOX5b-1 genes were downregulated (Fig 1C). 447 Interestingly, AtHCC2, which has also been implicated in ROS defence after UV-B light 448 exposure [38], responded to the chemical oxidative stressors mostly with downregulation.

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When antimycin A was applied, however, the AtHCC2 transcript levels were initially 450 reduced but increased after 6 h ( Fig 1C). These findings confirm previous reports on the 21 451 sensitivity of the oxidative defence machinery to the type of stressor and the time point of 452 analysis [37,46]. Taken together, this data supports that AtCOX11 likely has an auxiliary 453 role in the oxidative defence in addition to its main role in copper transport. 454 One would expect that knockdown and overexpression of an oxidative stress defence 455 protein to result in higher and lower ROS levels, respectively. Nevertheless, at first 456 glance, our experiments did not fulfil these predictions and even yielded opposite results 457 with knock-down plant mutants having reduced ROS levels (Fig 2). This reduction could inducing menadione (Fig 4B), showing that COX11 proteins are indeed able to reduce 495 oxidative stress. Since the antioxidative function was exerted even in the non-native 23 496 cellular environment, one may speculate that COX11 proteins are able to function in ROS 497 defence, independently of other proteins.

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The mutation of all three cysteines in the COX11 proteins mostly abolished their 499 ability to convey growth under oxidative stress, emphasizing the role of these amino acid 500 residues in ROS detoxification. However, when compared with the GFP-expressing 501 control strain, the triple cys mutants were still more resistant to menadione, hinting at an of 110 µM the loss of one of the three cysteines had no effect, but a mere increase of 516 10% to 120 µM made the difference in oxidative stress resistance readily apparent. COX11 revealed that all three conserved cysteines are on the protein surface and thus 528 easily accessible to oxidation by ROS molecules and S-S bridge formation. Of note is that 529 COX11 proteins -in addition to the formation of intramolecular disulphide bridges within 530 a single COX11 subunit -could potentially also form intermolecular bridges between two 531 COX11 subunits or between COX11 and another protein or e.g. glutathione (GSH).

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As an alternative explanation for their antioxidant activity, the COX11 copper 533 chaperones may use the bound copper to detoxify ROS. However, this scenario seems 534 unlikely, because our experiments demonstrate that the loss of one of the cysteines in 535 the copper-binding motif (cys 221 and 210 in Arabidopsis and yeast COX11, respectively) 536 did not eliminate the COX11 antioxidant activity (Fig 4B).

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Taken together, the findings that the mutation of the respective cysteines had the 538 same positive or negative antioxidant effects in two evolutionary distant organisms like 539 Arabidopsis and yeast, pinpoint that these cysteines and their functions were obviously 540 important to be conserved during evolution.

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Therefore, it seems plausible that the formation of either disulphide bridge a or c, or 542 both, is the mechanism by which COX11 proteins detoxify ROS. These potentially ROS-543 induced S-S bridges could subsequently be reduced in the IMS by thioredoxins or 544 proteins with a putative thioredoxin domain such as AtHCC2 [38], or by other redox 545 systems, e.g. the ERV1/MIA40 IMS protein import system [3]. While many open questions 546 remain regarding the role of COX11 proteins in ROS metabolism, the data presented here