PrrC, a Sco homologue from Rhodobacter sphaeroides, possesses thiol-disulfide oxidoreductase activity

FEBS Letters 581 (2007) 4663–4667

PrrC, a Sco homologue from Rhodobacter sphaeroides, possesses thiol-disulfide oxidoreductase activity Alison C. Badrick, Amanda J. Hamilton, Paul V. Bernhardt*, Christopher E. Jones, Ulrike Kappler, Michael P. Jennings, Alastair G. McEwan* Centre for Metals in Biology, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia Received 7 August 2007; accepted 23 August 2007 Available online 4 September 2007 Edited by Richard Cogdell

Abstract PrrC is a Sco homologue in Rhodobacter sphaeroides that is associated with PrrBA, a two-component signal transduction system that induces photosynthesis gene expression in response to a decrease in oxygen tension. Although Sco proteins have been shown to bind copper the observation that they are structurally-related to thioredoxins suggested that they might possess thiol-disulfide oxidoreductase activity. Our results show that PrrC reduces Cu2+ to Cu+ and possesses disulfide reductase activity. These results indicate that some bacterial Sco proteins may have biochemical properties that are distinct from those of mitochondrial Sco proteins.  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Sco; Copper; Cytochrome c oxidase; Thioredoxin; Insulin

1. Introduction Mitochondrial and bacterial synthesis of cytochrome oxidase (Sco) proteins are critical for the assembly of cytochrome aa3 in the respiratory electron transport chain [1]. Sco proteins and their homologues possess a highly conserved motif consisting of Cys-X-X-X-Cys-Pro, (X signifying a non-conserved residue). Structural characterisation of Sco proteins has shown that they possess a thioredoxin-fold consisting of a core of four b-sheets surrounded by three a-helices [2], and each Sco protein potentially binds one copper ion [3]. The putative copper-binding site comprises one histidine (His135, human Sco1 numbering), and the two cysteine residues (Cys45 and Cys49) from the conserved Cys-X-X-X-Cys-Pro motif [3]. *

Corresponding authors. Fax: +61 7 3365 4699 (A.G. McEwan); fax: +61 7 3365 4299 (P.V. Bernhardt). E-mail addresses: [email protected] (P.V. Bernhardt), mcewan @uq.edu.au (A.G. McEwan). Abbreviations: BC, bathocuproine 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline; BSA, bovine serum albumin; Co(trans-diammac), trans-6,13-dimethyl-(1,4,8,11-tetraazacyclotetradecane-6,13-diamine)cobalt(III) perchlorate; cPrrC, cleaved PrrC; DTNB, 5,5-dithiobis(2nitrobenzoic acid); DTT, dithiothreitol, (2R,3R)-1,4-bis-sulfanylbutane-2,3-diol; EPR, Electron Paramagnetic Resonance; htPrrC, 6·-his-tagged PrrC; IPTG, isopropyl-b-D -thiogalactopyranoside; oxPrrC, oxidised PrrC; PrrC, photosynthetic regulatory response; rPrrC, reduced PrrC; Sco, synthesis of cytochrome oxidase; Trx, thioredoxin

Structures of homologues from Bacillus subtilis (BsSco) [4], yeast (ySco1) [5] and human Sco1 (hSco1) [2,6] appear to show the location of metal ion incorporation. The high level of conservation of the metal-binding motif suggests a common copper-binding ability across a range of organisms [7]. The copper-binding ability of Sco is considered essential to its function. Cytochrome aa3 contains two different copper-binding sites, CuA (cytochrome aa3) and CuB; Sco is thought to be involved in donating copper to the CuA centre and thus it has a central role in cytochrome oxidase synthesis [2,4,5,7–10]. In addition to copper delivery there may be additional functions for Sco proteins. Photosynthetic regulatory response (PrrC) is a Sco homologue present in the purple photosynthetic bacterium Rhodobacter sphaeroides [11]. R. sphaeroides is a Gram-negative a-proteobacterium that performs anoxygenic photosynthesis [12] and is also capable of carrying out respiration under aerobic conditions. Although R. sphaeroides possesses a cytochrome oxidase with a CuA centre (cytochrome aa3), it also contains another cytochrome oxidase (cytochrome cbb3) which lacks a CuA centre. R. sphaeroides has a close relative, Rhodobacter capsulatus, which only possesses cytochrome cbb3 but still contains a Sco protein homologue (SenC) [13]. This situation has been discovered in a number of other bacteria including the genus Neisseria [14]. The presence of Sco in bacteria that possess a CuA-free cytochrome oxidase suggests that the Sco homologues in these organisms carry out an alternative function to copper delivery. In this context the strong similarity of the Sco structure to that of thioredoxin (Trx) [7] is intriguing. In the current work, we investigated the thiol redox activity and copper-binding ability of PrrC using complementary spectroscopic and electrochemical techniques.

2. Materials and methods 2.1. Materials All experiments were performed using sterile Milli-Q water (Millipore). Metal ion solutions were made up in HPLC-grade water (LabScan Analytical Sciences). trans-6,13-dimethyl-(1,4,8,11-tetraazacyclotetradecane-6,13-diamine) cobalt(III)perchlorate (Co(trans-diammac)) was previously synthesised [15]. 2.2. Expression and purification of PrrC Expression and purification of PrrC was essentially as described previously [16]. Briefly, the plasmid containing PrrC (pProEx-PrrC) was transformed into competent E. coli BL21(DE3) cells. 5 · 1 L flasks were inoculated from an overnight culture and allowed to grow to an OD600 of 0.6. Isopropyl-b-D -thiogalactopyranoside (IPTG)

0014-5793/$32.00  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.08.058

4664 (1 mM) was used to induce expression and the flasks were shaken at 30 C for 2.5 h. Cells were harvested and the cell pellet resuspended in lysis buffer (50 mM NaH2PO4, 200 mM NaCl, 10 mM imidazole). A Complete protease inhibitor cocktail tablet (Roche Diagnostics Australia, Castle Hill, Australia) and excess DNase from bovine pancreas (Sigma, St. Louis, MO) were added, and cells were passed through a French pressure cell press (American Instrument Company, Silver Springs, MD). Cellular debris was removed by centrifugation, and supernatants were mixed with a Chelating SepharoseTM Fast Flow column gel (GE Healthcare, Buckinghamshire, UK), charged with 0.1 M NiSO4. Fifteen column volumes wash buffer (50 mM NaH2PO4, 200 mM NaCl, 20 mM imidazole) were passed through the column to elute unbound (non-tagged) proteins. Eight column volumes of elution buffer (50 mM NaH2PO4, 200 mM NaCl, 250 mM imidazole) were added to elute 6·-his-tagged PrrC (htPrrC). Column fractions containing htPrrC were pooled and stored at 80 C. SDS–PAGE and MALDI-MS (Voyager-DETM STR BioSpectrometryTM Workstation (Applied Biosystems)) were used to confirm the presence of purified htPrrC. AcTEVTM Protease (Invitrogen) was used to cleave the 6·-his tag from htPrrC under conditions recommended by the manufacturer. Cleavage was complete after 40 h at 4 C. Cleaved PrrC (cPrrC) was separated from the AcTEV protease using Chelating Sepharose Fast Flow chromatography. Atomic absorption spectroscopy (SpectrAA300, Varian Techtron Pty. Ltd., Victoria) showed the absence of metals in the purified protein. cPrrC was stored at 80 C for further experiments. 2.3. Copper-binding assays PrrC was reduced (rPrrC) using excess dithiothreitol (DTT) and heating at 37 C for 2 h. In an anaerobic chamber, rPrrC was separated from DTT using a PD-10 column equilibrated with 50 mM MES hydrate, pH 6.0. Full reduction of all disulfide bonds in PrrC was confirmed using the 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) assay [18]. Small aliquots of CuCl2 Æ 2H2O were added from a 1 mM solution to rPrrC in a 1 cm path-length cuvette, and changes to the spectra were plotted as a function of added metal. All UV/Vis spectra were recorded on a Hitachi U-3000 spectrophotometer. In a separate experiment, the presence of Cu+ in the Cu2+/PrrC solution was determined by the addition of bathocuproine (BC, Biochimika). The [CuI(BC)2]+ complex has a characteristic absorbance at 485 nm and e = 12 250 M 1 cm 1. Copper(I) titrations were also performed using freshly prepared 20 mM Cu(I)(CH3CN)4 Æ PF6 (Aldrich) in 100% acetonitrile. 2.4. Electron paramagnetic resonance (EPR) analysis EPR measurements were undertaken at 140 K using a Bruker Elexsys Electron Paramagnetic Resonance E580 instrument (X-band, 9.3 GHz). Temperature was controlled by an ER4131VT unit and all spectra were collected with 100 kHz modulation frequency at a power of 6.331 mW under the control of the Bruker Xepr software. Spectra were baseline-corrected using polynomial functions and smoothed using Fourier filtering available in Xepr. 2.5. Cyclic voltammetry Measurements were carried out using a BAS100B/W potentiostat and BAS C3 Cell Stand. The working electrode was a glassy carbon disk, the counter electrode was a Pt wire and a Ag/AgCl (3 M NaCl) reference electrode was used. The potential was swept between 0.3 V and 1 V vs. Ag/AgCl, with a sweep rate of 2 mV/s. For each analysis, the working electrode was polished with alumina and soaked in deoxygenated Tris–HCl (50 mM, pH 8.0). 2.6. Turbidimetric assay of insulin disulfide reduction The PrrC-catalysed reduction of insulin disulfides was monitored using the method of Holmgren [17]. A reaction mixture comprising 3 lM protein and 2.5 mM DTT was incubated at room temperature for 15 min in 50 mM K2HPO4, pH 8.0. Insulin was added to the solution to a final concentration of 0.17 mM, and the volume made up to 600 lL with buffer. The precipitation of reduced insulin chains was monitored turbidimetrically at 650 nm for up to 120 min. E. coli Trx and bovine serum albumin (BSA) (each at 3 lM) were used as positive and negative controls respectively. A sample containing only 2.5 mM DTT (no protein) was also tested as a control.

A.C. Badrick et al. / FEBS Letters 581 (2007) 4663–4667

3. Results 3.1. Binding of copper to PrrC The binding of copper to rPrrC was monitored using UV/Vis spectroscopy. Fig. 1 shows that titration of Cu2+ into rPrrC resulted in an increase in absorption at 270 nm and the appearance of a band at 400 nm. The absorption at 270 nm did not plateau, whilst the 400 nm transition reached maximum amplitude after 0.5 equivalent of copper had been added (Fig. 1, inset). Many copper related peaks occur near 270 nm including Cu2+–N charge-transfer transitions. The non-linearity of the increasing 270 nm peak may result from transitions that change as the titration proceeds, whereas the peak at 400 nm may result from a single contributing species. Indeed, Cu2+ titrated into oxidised PrrC (oxPrrC) resulted in a peak around 270 nm, but no 400 nm peak (data not shown). The 400 nm transition plateau at 0.5 mole equivalent copper is suggestive of a metal-induced dimer. However, native gel electrophoresis suggested the protein was monomeric, consistent with previous results [16]. There were no transitions in the visible region. Some studies have shown that Sco protein can bind Cu(I). The titration of reduced cPrrC with Cu+ is shown in Fig. S1. An increase in absorbance was observed at around 260 nm but this did not plateau. The 260 nm peak is suggestive of S(Cys)–Cu+ charge transfer, however PrrC contains four cysteines and the absorbance observed at two equivalents of Cu+ suggests all four are binding Cu+ (e = 5000 M 1 cm 1/Cu–S bond, Fig. S1). This result suggests that Cu+ was not specifically binding to the C-X-X-X-C site implicated in other Sco proteins. 3.2. rPrrC reduces Cu2+ Previous work has shown that one pair of thiols in rPrrC is extremely sensitive to oxidation. These thiols were suggested to be involved in copper-binding and it was the sensitivity of these thiols to oxidation that prevented direct titration of Cu2+ [16]. In order to determine if the sensitivity of these thiols towards oxidation affected copper-binding under our conditions, we used EPR to directly monitor Cu2+. When Cu2+ is added to MES buffer, a spectrum characteristic of an axially symmetric Cu2+ centre is produced (Fig. 2, dashed line). When Cu2+, identical in concentration to that added to MES (15 lM), was added to rPrrC the spectrum obtained is entirely different (Fig. 2, solid line). The intensity of the g^ transition was reduced by approximately 80%, compared with that of copper in MES buffer, and the hyperfine couplings are broadened. The broadening prevented accurate determination of gi and Ai. However, consistent with the absorption data (Fig. 1) the presence of an EPR signal indicated that some Cu2+ was bound to PrrC. To investigate whether the loss of the Cu2+ intensity was as a result of reduction to EPR-silent Cu+ we used the Cu+-specific chelator bathocuproine (BC). In a separate experiment Cu2+ was added to rPrrC, then, after 5 min, BC was added. Fig. 2, inset, shows the characteristic absorbance at 485 nm due to Cu+ bound BC. Taken together these data suggest that PrrC can mediate the reduction of Cu2+. 3.3. rPrrC exhibits thioredoxin-like activity – electrochemical evidence Sco proteins and homologues appear to be structurally similar to thioredoxins and peroxiredoxins [16], yet none have

A.C. Badrick et al. / FEBS Letters 581 (2007) 4663–4667

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Wavelength (nm) Fig. 1. UV/Vis absorption spectroscopy of copper(II) additions to cPrrC. Reduced cPrrC (2.3 lM) was titrated with Cu2+, up to 1.3 Cu2+ equivalents (in 0.5 lM increments), and the spectra collected between 220 nm and 800 nm. Inset shows absorption at 400 nm as a function of the Cu2+ equivalents added. All spectra have the apo spectrum subtracted.

trode and acted as an artificial electron donor to maintain htPrrC in a reduced state. Only after the addition of NADH was catalysis observed (Fig. 3A, solid line). Steady state catalysis was inferred from the sigmoidal steady-state voltammogram obtained, where the reduced form of the mediator was continually regenerated at low potential (less than 800 mV) in order to continue to reduce NADH. When only the mediator was added to donate electrons to the protein, no catalytic current was observed, and only a diffusion-controlled (peakshaped) voltammogram was observed (see Fig. 3A, dashed line). A cyclic voltammogram of a solution containing only NADH and Co(trans-diammac) gave the same result (Fig. 3A, dashed line). This voltammogram is again characteristic of the non-catalytic reversible reduction and re-oxidation of Co(trans-diammac). Voltammograms of combinations of NADH, insulin and rPrrC do not show any catalytic current (Supplementary material, Fig. S2). The transfer of electrons in this experiment is shown schematically in Fig. 3B; the mediator was required to reduce NAD+ to NADH (as direct reduction of NAD+ at a glassy carbon electrode is typically sluggish [19]). NADH, formed electrochemically, then reduces PrrC. The ensuing protein rPrrC can act on disulfide-bonded proteins such as insulin.

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Fig. 2. PrrC reduces Cu . X-band EPR spectra obtained after the addition of Cu2+ (15 lM) to MES buffer (dashed line) or reduced PrrC (15 lM, dashed line). Inset shows the UV/Vis absorption spectra obtained after addition of bathocuproine to a solution of reduced PrrC (15 lM). Absorption at 485 nm is due to Cu+ bound to bathocuproine.

been shown to have thiol-disulfide oxidoreductase activity. Due to the effectiveness of rPrrC in reducing Cu2+, we investigated the ability of this protein to act as a thioredoxin-like protein. Initially, we investigated thioredoxin activity using cyclic voltammetry to monitor the catalytic reduction of insulin. Co(trans-diammac) [15] was used as an electron transfer mediator; the mediator was reduced to CoII(trans-diammac) at the elec-

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Fig. 3. Cyclic voltammetric analysis of the PrrC-mediated reduction of insulin. (A) Voltammogram of Co(trans-diammac) (0.1 mM), NADH (0.1 mM) and insulin (1 mg/mL) showing classic transient voltammetry of Co(trans-diammac) (dashed line) and the voltammogram of Co(trans-diammac) (0.1 mM), NADH (0.1 mM), PrrC (1.3 lM) and insulin (1 mg/mL) showing steady state catalytic reductive voltammetry (solid line). All experiments were conducted with a sweep-rate of 2 mV/s in a buffer composed of 40 mM Tris–HCl and 19 mM NaCl, pH 8. (B) Scheme showing proposed mechanism for the mediated reduction of insulin involving Co(trans-diammac) as the electroactive species.

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A.C. Badrick et al. / FEBS Letters 581 (2007) 4663–4667

3.4. PrrC exhibits thioredoxin-like activity – biochemical evidence To support our voltammetric analysis, we investigated the PrrC-enhanced reduction of insulin in the presence of DTT [17]. Fig. 4 shows that E. coli thioredoxin (Trx) can enhance the reduction of insulin, compared with control samples (DTT alone or with BSA). When rPrrC was reacted with insulin, reduction was about ten-fold slower compared with Trx, and there was a longer lag time (28 min). However, these data show that whilst not as kinetically effective as Trx, PrrC is able to mediate the reduction of insulin disulfide bonds. The rate calculated for Trx in this experiment (0.0513 DA650 nm/min) is comparable with that obtained by Holmgren [17]. Insulin reduction was also catalysed in the presence of a Sco homologue isolated from Neisseria gonorrhoeae (Hamilton, McEwan and Jennings unpublished observation).

4. Discussion Although there has been substantial progress toward an understanding of the structure of Sco proteins, information about their biochemical properties that could lead to an understanding of their mechanism of action is still incomplete. Human Sco1 and Sco2 have been shown have distinct properties, consistent with the evidence that sco1 and sco2 are paralogs [20]. It has been demonstrated that hSco2 purified from E. coli can bind copper [21]. However, copper metallation of hSco1 expressed in yeast is dependent upon the co-expression of Cox17, the copper chaperone that delivers copper to the inter-membrane space of the mitochondrion [3]. Using UV/Vis and EPR spectroscopy we observed that rPrrC was able to bind some Cu2+; potentially this involves thiolate ligands since a similar titration using oxidised PrrC did not

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Time (min) Fig. 4. Insulin reductase activity of PrrC. PrrC (3 lM, 0.1 mg/mL) was compared with E. coli Trx (3 lM), BSA (3 lM) and DTT (2.5 mM). The precipitation of insulin was monitored at 650 nm [25].

generate a 400 nm absorption band. Although our data are suggestive of some Cu2+-thiolate coordination, we cannot be sure about the nature of the complex as it is unlikely to be similar to previously observed copper-Sco complexes. For instance, copper-loaded hSco1 has a number of electronic transitions (at 360, 480 and 610 nm), some of which are attributed to S(Cys)-to-Cu charge-transfer transitions [3]. Identical absorption bands were also observed in BsSco [7], suggesting the nature of the copper complex is conserved in these Sco proteins. In the presence of a stoichiometric quantity of Cu2+, rPrrC was oxidised and Cu2+ was reduced to Cu+ (Fig. 2). This suggests that the failure to observe formation of a stable copper centre in PrrC may be a consequence of its redox activity towards Cu2+. This conclusion is supported by our observation that PrrC did not form a stable stoichiometric complex with Cu+. Although it is recognised as being related to thioredoxins, hSco1 does not appear to possess thiol-sulfide oxidoreductase activity [6]. In contrast, the disulfide reductase activity of PrrC and Neisseria Sco (Hamilton, McEwan and Jennings unpublished observation) indicates that at least two bacterial Sco proteins can act as periplasmic thiol-disulfide oxidoreductases. Divalent copper is able to oxidise dithiol groups and cause the formation of intermolecular disulfide bonds between protein molecules and consequent oligomerisation of the protein. DsbC has been shown to restore these proteins to their native disulfide-bound forms and thus may be important in protection of the cell against oxidative stress [22]. Consequently, a role for bacterial Sco proteins may be to contribute to protection of periplasmic proteins against copper-induced disruption of protein disulfide bridge formation and folding. In this context, it is interesting that a Neisseria Sco mutant is sensitive to viologen (paraquat)-induced oxidative stress [14]. PrrC from R. sphaeroides may have a more specialised function, since it is associated with the PrrB/PrrA two-component signal transduction system. Mutation of prrC results in increased photosynthetic gene expression in the presence of high oxygen, suggesting that PrrC is required for the correct functioning of the sensor kinase/phosphatase PrrB [11,23]. In the homologous system in R. capsulatus it has been observed that inactivation of the sensor kinase RegB involves formation of an intramolecular disulfide bridge involving a single cysteine residue. It has also been demonstrated that RegB binds a single copper ion but the way in which this is connected to the redox activity of the cysteine residue is not clear [24]. Although the mechanism is unknown, these data suggest that the thioredoxin-like activity of PrrC may be central to the regulation of PrrB (RegB) that occurs upon a reduction in oxygen tension. A signalling role for mitochondrial Sco proteins has also been postulated by Williams and co-workers [6], although the route of signal transduction has not been defined. However, our new observations indicate that there may be significant diversity of activity of Sco proteins towards copper ions and thiols/disulfides and this may be linked to differences in biological function. Acknowledgements: The National Health and Medical Research Council (Australia) is acknowledged for providing support via a Program Grant (284214) to AGM and MPJ and a CJ Martin Fellowship (252972) to CEJ. AJH is the recipient of an Australian Postgraduate Award. PVB acknowledges financial support from the Australian Research Council and the University of Queensland.

A.C. Badrick et al. / FEBS Letters 581 (2007) 4663–4667

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