Article pubs.acs.org/biochemistry
Universal Stress Protein Regulates Electron Transfer and Superoxide Generation Activities of the Cytochrome bc1 Complex from Rhodobacter sphaeroides Ting Su,# Qiyu Wang,# Linda Yu, and Chang-An Yu* Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, United States S Supporting Information *
ABSTRACT: Interactions between Rhodobacter sphaeroides cytochrome bc1 complex (Rsbc1) and soluble cytosolic proteins were studied by a precipitation pull-down technique. After being purified, detergent-dispersed Rsbc1 complex was incubated with soluble cytosolic fraction and then dialyzed in the absence of detergent; the interacting proteins were coprecipitated with Rsbc1 complex upon centrifugation. One of the cytosolic proteins pulled down by Rsbc1 complex was identified by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS) to be the reported R. sphaeroides universal stress protein (UspA). Incubating purified UspA with the detergent dispersed bc1 complex resulted in an increase in the Rsbc1 complex activity by 60% and a decrease in superoxide generation activity by the complex by more than 70%. These UspA effects were only observed with Rsbc1 complexes containing subunit IV and assayed under aerobic conditions. These results suggest that the interaction between UspA and Rsbc1 complex may play an important role in R. sphaeroides cells during oxidative stress. Using a biotin label transfer technique, cytochrome c1 of the Rsbc1 complex was identified as the interacting site for UspA.
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soluble/dispersed in a buffer containing detergents. Upon removal of detergent by dialysis, mbc1 becomes turbid and is recovered in the precipitate after centrifugation. Therefore, it is expected that a matrix protein that interacts with mbc1 will coprecipitated with mbc1 upon dialysis. By using this precipitation pull-down method, mitochondrial malate dehydrogenase has been identified as a protein interacting with mbc1.5 The interacting subunits of the mitochondrial bc1 complex have been identified as core I, core II, and ISP.5 Since Rsbc1 also possesses the same soluble/insoluble properties as that of the mitochondrial complex, we used this technique to search for proteins that interact with Rsbc1 in the soluble cytoplasmic protein fraction of this bacterium.5 One of the cytosolic proteins pulled down by the Rsbc1 complex was identified as the reported R. sphaeroides universal stress protein (UspA)7 by liquid chromatography-coupled tandem mass spectrometry (LC/MS/MS).
he cytochrome bc1 complex from the photosynthetic bacterium Rhodobacter sphaeroides catalyzes the electron transfer from ubiquinol to cytochrome c21 and translocates protons across the membrane to generate a pH gradient and membrane potential for ATP synthesis. There are four protein subunits in this bacterial complex with apparent molecular masses of 43, 31, 23, and 15 kDa. The three larger subunits are cytochrome b, housing two b-type hemes (b562 and b566), cytochrome c1, having one c-type heme (c1), and the iron− sulfur protein (ISP), containing a [2Fe−2S] cluster.1,2 The smallest subunit, subunit IV, containing no redox prosthetic group, is a supernumerary subunit.3,4 Although the structure and function of the R. sphaeroides cytochrome bc1 complex (Rsbc1) have been intensively studied, no attention has yet been dedicated to the interaction between Rsbc1 and the soluble cytoplasmic proteins. Recently, a precipitation pull-down method has been developed for the study of the interactions between mitochondrial matrix protein(s) and mitochondrial bc1 complex (mbc1).5 This method is based on the soluble/insoluble properties of mbc1 in the presence or absence of a detergent. Purified mbc1 is only © XXXX American Chemical Society
Received: June 14, 2015 Revised: November 17, 2015
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DOI: 10.1021/acs.biochem.5b00658 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry Universal stress protein in Escherichia coli K-12 (UspA) was first named by Nyström and Neidhardt.6 So far, more than 1000 different proteins that contain the UspA domain (Pfam accession number PF0582) have been detected.7 The UspA domain may be present individually or fused to another domain.8 About 80% of the UspA proteins in bacteria and archaea have only a single UspA domain or two found in tandem.9 The crystal structures of UspA from Haemophilus inf luenza10 and Methanococcus jannaschii MJ057711 have been determined. The atomic structures show that they are asymmetric dimers containing similar α/β tertiary folds. UspA is induced by many stresses such as glucose or phosphate starvation, heat shock, or oxidative stress.12 However, UspA is not induced by cold shock.12 UspA is needed for cells to protect DNA from damage.13,14 UspA inactivation can induce a longer lag when cells at stationary-phase13−15 are inoculated in fresh medium. Nachin et al.16 also found that UspA from E. coli plays an important role in oxidative defense. During exponential growth, UspA can protect the cells against superoxide damage.14,17 Although there has been progress in the study of the UspA family of proteins, their biochemical activities and the mechanisms are still not fully understood. Herein, we report the identification of UspA as one of the soluble cytoplasmic proteins that interacts with Rsbc1. The effect on the electron transfer and superoxide production activities of the Rsbc1 complex as well as the likely interaction subunits in Rsbc1 was investigated.
precipitate was collected, mixed with 1 mL of 75% ethanol, and centrifuged at 7500g for 5 min. The pellets were collected, mixed with 1 mL ethanol, and centrifuged at 14000g for 5 min. The precipitate was collected, air-dried, and dissolved in 30 μL of the diethylpyrocarbonate (DEPC) treated, RNase-free water. RT-PCR. In a tube containing 1 μg of RNA and 0.5 μg of the random primers was heated to 70 °C for 5 min, cooled immediately on ice, and then mixed gently with M-MLV 5X reaction buffer, dNTPs, recombinant RNasin ribonuclease inhibitor, and Moloney murine leukemia virus reverse transcriptase (M-MLV RT). The mixture was incubated at 37 °C for 60 min for the synthesis of cDNA. After the R. sphaeroides cDNA library was obtained, the following primers were used for PCR amplification of the gene for UspA. The 6xHis tag was added to the C-terminal end of the proteins. Forward primer: 5′ TAA GAA GGA GAT ATA CC ATG GCC TAT AAA TCC TTG 3′, Reverse primer: 5′ GTG ATG GTG GTG ATG ATG GTG CGC CAT CAG GAC GGG 3′ Protein Expression and Purification. In order to ligate the UspA gene into the expression vector, pET-28a(+), the Xba I and Not I sites were introduced to the DNA fragment containing the UspA gene. This was done by PCR amplification using these two primers (forward 5′ TGC TCT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CC 3′; reverse 5′ TTT TCC TTT TGC GGC CGC TCA GTG ATG GTG GTG ATG ATG 3′). The amplified DNA fragment was then digested with XbaI and NotI. The XbaI−NotI fragment containing the UspA gene was ligated into the XbaI and NotI sites of an expression vector pET-28a(+) to generate the UspA expression vector (pET-UspA). The E. coli cells harboring pET-UspA were grown at 37 °C in LB medium. After the OD reached 0.8, isopropyl β-D-1thiogalactopyranoside (IPTG) (1 mM) was added, and the culture was continued to be grown for 4 h. The cells were harvested by centrifugation and broken by sonification at 4 °C intermittently for a total of 16 min, 5 s on and 5 s off. The broken cell suspension was centrifuged at 23000g for 10 min, and the supernatant obtained was applied onto a Ni-NTA column. The column was washed with PBS buffer containing 20 mM histidine and then eluted with PBS buffer containing 200 mM histidine. The fractions containing UspA protein were combined, dialyzed against PBS buffer, and concentrated using polyethylene glycol (PEG) 8000. The Pulldown Experiment. Seven microliters of purified Rsbc1 (cyt b, 650 μM) was incubated with 500 μL of the soluble cytoplasmic protein fraction (2 μg/μL) or with 100 μL of purified, recombinant UspA (0.7 μg/μL) on ice for about 40 min. After incubation, the mixture was added to PBS buffer to a final volume of 0.5 mL, dialyzed against the PBS buffer at 4 °C for 3 h, and then centrifuged at 104000g for 40 min. The precipitates thus obtained were dissolved in 50 mM Tris-HCl buffer, pH 8.0 at 4 °C, containing 200 mM NaCl, and 0.01% DM. Mass Spectroscopy. Samples were subjected to SDSPAGE followed by staining with Coomassie blue. The selected protein bands were sliced from the gel, then washed with 50% acetonitrile, and then with 50 mM ammonium bicarbonate, pH 8.0. The washed gel slices were dehydrated by 100% acetonitrile and air-dried briefly. The dried gel slices were first incubated with a reducing buffer containing 10 mM tris (2carboxyethyl)phosphine (TCEP) and 50 mM ammonium bicarbonate for 1 h at room temperature and then with an alkylating buffer containing 50 mM ammonium bicarbonate,
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EXPERIMENTAL PROCEDURES Materials. Ni-NTA gel and QiAprep spin Miniprep Kit were purchased from Sigma. N-Dodecyl-ß-D-maltopyranoside (DM) and N-octyl-ß-D-glucopyranoside (OG) were purchased from Anatrace. Cross-linker sulfo-SBED (sulfosuccinimidyl-2[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl1,3-dithiopropionate) was purchased from Pierce. Antimycin was from fluka. 2,3-Dimethoxy-5-methyl-6-(10-bromodecyl)1,4-benzoquinol (Q0C10BrH2) was prepared as previously reported.18 Preparation of Soluble Cytoplasmic Protein Fraction from R. sphaeroides Cells. The wild type R. sphaeroides cells were suspended in 20 mM Tris-succinate buffer, pH 8.0, and passed through a French pressure cell. The broken cell suspensions were centrifuged at 23000g for 30 min, and the precipitates were discarded. The supernatant was centrifuged again at 218000g for 2.5 h to remove chromatophore. The supernatant thus obtained (the soluble cytoplasmic protein fraction) was dialyzed against PBS buffer for 3 h and then centrifuged at 104000g for 30 min to remove precipitates formed, if any. Preparation of Purified, Recombinant UspA. Purified, recombinant UspA was prepared by a procedure involving three steps: (a) RNA extraction, (b) RT-PCR, and (c) protein expression and purification. These three steps were described as follows: RNA Extraction. A total of 500 μL of R. sphaeroides cells was centrifuged at 6000g for 5 min at 4 °C. The supernatant was discarded, and the precipitate was suspended with 1 mL of Trizol and incubated at room temperature for 5 min before 200 μL of cold chloroform was added. The mixture was incubated for 3 min and then centrifuged at 12000g for 15 min. The colorless upper phase containing RNA (about 400 μL) was transferred to a new tube, 500 μL of isopropanol was added, and the sample was centrifuged at 15000g for 10 min. The B
DOI: 10.1021/acs.biochem.5b00658 Biochemistry XXXX, XXX, XXX−XXX
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reaction analyzer SX.18 MV (Leatherhead, England), while leaving the excitation light off and registering light emission.22 The amount of chemiluminescence produced is quantified by the mV read-out from the instrument. Reactions were carried out at 23 °C by mixing solutions A and B in a 1:1 ratio. Solution A contains 100 mM phosphate buffer, pH 7.4, 0.01% DM, 1 mM NaN3, and 0.5 μM Rsbc1 complex or 0.9 μM xanthine oxidase. Solution B contains 50 μM QH2, or hypoxanthine and 4 μM MCLA, in water.
pH 8.0 and 55 mM iodoacetamide in the dark for 1 h at room temperature. In gel digestion was carried out with 8 μg/mL freshly prepared trypsin in 50 mM ammonium bicarbonate, pH 8.0 at 37 °C overnight. After digestion, 0.5% TFA was used to extract the tryptic peptide products, which were used for subsequent analysis by LC-MS/MS. The mass spectrometric analyses were performed in the DNA/protein Resource Facility of Oklahoma State University. Cytochrome bc1 Complexes Preparation and Activity Assay. The wild-type complex19 and mutant complexes of S287R(cytb)/V135S(ISP), 20 c 1 -14Gly-IV-6His, 19 ΔIV, 21 H198N,22 and H111N22 were prepared as previously reported. The concentrations of cytochromes b and c1 were determined spectrophotometrically using millimolar extinction coefficients of 28.522 and 17.5 cm−1 mM−1,22 respectively. For activity determination, the Rsbc1 complexes were diluted with 20 mM Tris-HCl buffer, pH 8.0, containing 0.01% DM to a final concentration of cytochrome b of 1 μM. Appropriate amounts of the diluted proteins were added to 1 mL of assay mixture containing 100 mM Na+/K+ phosphate buffer, pH 7.4, 1 mM EDTA, 50 μM cytochrome c, and 25 μM Q0C10BrH2. The reduction of cytochrome c was measured by the increase of the absorbance at 550 nm wavelength in a Shimadzu UV-2401 PC spectrophotometer at 23 °C. A millimolar extinction coefficient of 18.5 was used for calculation of the activity. Cross-Linking. A heterobifunctional chemical cross-linker, sulfo-SBED, containing a sulfonated N-hydroxysuccinimide (sulfo-NHS) active ester, a biotin, and a photoactivatable aryl azide was used. A total of 1.5 μL of sulfo-SBED (50 mM) was added to 120 μL of purified UspA (27 μM), and the mixture was incubated on ice for 2 h. This step was for sulfo-SBED to label UspA through the reaction of the amine-reactive sulfoNHS ester with the N-terminus of UspA and its side chains of lysine residues. After incubation, 100 μL of PBS buffer was added, and the sample was incubated at room temperature for another 30 min before being subjected to dialysis against 1 L of PBS buffer for 3 h with two changes of buffer. This step is to remove unreacted (excess) cross-linker. The dialyzed sample was centrifuged at 66000g for 30 min to remove the denatured protein formed, if any. After dialysis to remove unreacted crosslinker and centrifugation to remove denatured UspA, the labeled UspA was mixed with Rsbc1. The mixture was illuminated with a long wave UV lamp (365 nm), at a distance of 5 cm for 15 min. The disulfide bond in the spacer arm originally attached to the Sulfo-NHS ester was cleaved by incubating it with 100 mM ß-mercaptoethanol. The final biotinlabeled proteins were subjected to Western blotting analysis. Gel Electrophoresis and Western Blot. The SDS-PAGE was performed with a Bio-Rad Mini-Protein dual slab vertical cell. Samples were treated with a sample buffer containing 10 mM Tris-HCl buffer, pH 6.8, 1% SDS, 3% glycerol, and 0.4% ßmercaptoethanol for 10 min at room temperature before electrophoresis. The polypeptides on SDS-PAGE gel were transferred to a 45-μm nitrocellulose membrane by immunoblotting at 16 V for 60 min and probed with antibodies against R. sphaeroides cytochrome b, c1, ISP, and subunit IV. Horseradish peroxidease (HRP) conjugated protein A was used as the secondary antibody. Color development was carried out by using HRP color development solution from Bio-Rad. Determination of Superoxide Generation. The production of superoxide anion by Rsbc1 or xanthine oxidase was determined by measuring the chemiluminescence of the MCLA-O2·− adduct in an Applied Photophysics Stopped-flow
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RESULTS AND DISCUSSION Identification of UspA as an Interacting Protein with the Rsbc1 Complex. To identify the proteins that interact with Rsbc1, the coprecipitation pull-down method was applied here with the soluble cytoplasmic fraction of R. Sphaeroides.5 The detergent (0.5% OG) dispersed Rsbc1 was incubated with the soluble cytoplasmic protein fraction of R. sphaeroides at 4 °C for 40 min. After incubation, the mixture was dialyzed against PBS buffer to remove the detergent and then centrifuged to collect the precipitates. Figure 1A shows SDSPAGE of the recovered precipitates. In addition to the four subunits of Rsbc1 of which the molecular masses are 43, 31, 23, and 15 kDa, one clear band with molecular mass of 30 kDa was observed. This band were cut out from the gel, subjected to ingel digestion with trypsin, and analyzed by LC/MS/MS. It was identified as R. sphaeroides universal stress protein (UspA). To study the interaction between UspA and Rsbc1, we expressed and purified recombinant UspA with Ni-NTA column. We first obtained the UspA gene by RT-PCR with R. sphaeroides RNA extract as template. Then it was ligated into the pET-28a expression vector, and a 6-His tag was added at the C-terminal end. When a given amount of purified Rsbc1 was incubated with UspA for 40 min at 4 °C and then subjected to dialysis and centrifugation, UspA was coprecipitated with Rsbc1 in an amount similar to that of cytochrome c1 or ISP in the complex (see Figure 1B), judging by the color intensity of Coomassie blue stained protein bands in the SDS-PAGE. This result indicates that UspA is the protein that indeed interacts with Rsbc1. As an interacting protein for Rsbc1, it would be expected to exert changes in Rsbc1 activity upon interaction. The Rsbc1 activity increased by 60% upon the addition of UspA. To exclude the involvement of other small molecules from the purified UspA preparation in the activity increase, the UspA protein was denatured by heating up to 100 °C for 3 min before incubating with Rsbc1 complex. The denatured UspA shows no effect on Rsbc1, indicating that UspA is the one responsible for activity increase of Rsbc1. Nature of Interaction between UspA and Rsbc1. To investigate the nature of the interaction between UspA and Rsbc1, concentration dependency was examined. When a given concentration of purified Rsbc1 complex was incubated with varying concentrations of UspA, the electron transfer activity of the complex increased as the concentration of UspA in the incubation mixture increased. A maximum activity increase of 60% was observed when 2 mol of UspA were added per mole of Rsbc1 complex (see Figure 2). Since the titration curve for the increase in Rsbc1 activity by UspA shows saturation (see Figure 2), the interaction between UspA and Rsbc1 is specific. The effect of UspA on the Rsbc1 complex activity was also found to be incubation time dependent with 30−60 min at 4 °C being optimal. Although a 1:1 ratio was obtained, if we extrapolate the C
DOI: 10.1021/acs.biochem.5b00658 Biochemistry XXXX, XXX, XXX−XXX
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Figure 2. Effect of UspA concentration on the Rsbc1 complex activity. Various amounts of UspA were added to a given amount of bc1 complex to give the indicated molar ratios. After the mixtures were incubated on ice for 40 min, the Rsbc1 activity was determined. The activity of Rsbc1 complex only (2.5 μmol c red/min/nmol b) was used as 100%. N = 3, data are the means ± SE.
S287 in cytochrome b and V135 in ISP were replaced with R and S, respectively; the H111N, in which heme bH is lacking;22 and the H198N, in which heme bL is lacking.22 Addition of UspA to mutant complexes of ΔIV, H111N, and H198N, which have very low Rsbc1 activity (see Table 1), showed no increase in Rsbc1 activity. On the other hand, addition of UspA to mutant complexes c1-14Gly-IV-His and S287R (cyt b)/ V135S(ISP), which have high Rsbc1 activity, showed a 40% increase in activity (1:1 molar ratio), the same as that observed for the wild-type complex. These results indicate that UspA can only enhance Rsbc1 activity in the structurally intact complex. The subunit IV in Rsbc1 is required for the interaction with UspA, since addition of UspA to the ΔIV mutant complex shows no activity increase, while addition of UspA to the wildtype or the subunit IV fused mutant complex (c1-14Gly-IV-His) increased their Rsbc1 activities. Effect of UspA on Superoxide Generation by Rsbc1 Complex. It has been reported16 that UspA is expressed in cells during oxidative stress and the Rsbc1 complex is one of the major sites that produce superoxide in cells. Therefore, it is interesting to see whether or not UspA affects the superoxide production by the Rsbc1 complex. Superoxide production was determined by measuring the chemiluminescence of MCLAO2·− adduct in an Applied Photophysics stopped flow reaction analyzer SX.18MV-R by leaving the excitation light off and registering light emission.5 The chemiluminescence is expressed in mV in this system. Figure 3A shows the direct tracings of superoxide production by Rsbc1 in the presence of varying concentrations of UspA. The MCLA-O2.− chemiluminescence induced by Rsbc1 in the absence of UspA reached its maximum peak height of about 110 mV. After addition of increasing amounts of UspA to the complex, the chemiluminescence peak height decreased progressively. The minimum peak intensity of about 30 mV was reached when four folds UspA were added to the system. These results indicate that interaction between UspA and Rsbc1 decreases superoxide production by the Rsbc1 complex. Since antimycin A (AA) inhibited Rsbc1 complex produces more superoxide than uninhibited complex, we examine whether AA induced superoxide production could also be decreased by UspA. Figure 3B shows the concentration dependent on UspA on superoxide generation by the AA
Figure 1. Identification of UspA as an interacting protein with Rsbc1. (A) SDS PAGE of recovered precipitates. Lanes 1−3 were precipitates obtained from bc1 alone, the soluble cytoplasmic protein fraction alone, and Rsbc1 incubated with the soluble cytoplasmic protein fraction, after dialysis and centrifugation, respectively. In lane 3, the solid arrow indicates the major soluble cytoplasmic proteins pulled down by the wt Rsbc1. The stars represent nonspecific proteins in the precipitate. (B) SDS-PAGE of supernatant and precipitate recovered after dialysis and centrifugation of an incubating mixture of Rsbc1 and purified UspA. Seven microliter of Rsbc1 (650 μM of cyt b) was incubated with 100 μL of purified UspA (0.7 μg/μL) on ice for about 40 min. After incubation, the mixture was added PBS buffer to give the final volume of 0.5 mL. The mixtures were dialyzed against PBS buffer to remove the detergent. The samples were then centrifuged at 104000g for 30 min. The precipitates were collected and dissolved in 50 μL of 50 mM Tris-HCl, pH 8.0 buffer at 4 °C containing 200 mM NaCl, 0.01% DM, and 1% SDS. SUP represents supernatant, and PPT represents precipitates.
titration data, the need of a higher than 1:1 ratio to obtain the maximum activity enhancement requires discussion. There are several possibly explanations: (1) because of the low binding efficiency of UspA under the experimental condition; (2) the purified recombinant UspA may not be in fully active form; (3) UspA may function as a dimer. Effect of UspA on the Electron Transfer Activity of Various bc1 Complex Preparations. To further study the interaction between UspA and Rsbc1, the effect of UspA on the activity of various preparations of Rsbc1 complexes was examined (Table 1). These complexes include the ΔIV, in which subunit IV is lacking;21 the c1-14Gly-IV-6His, in which the N-terminus of subunit IV is fused into the C-terminus of cytochrome c1;19 the S287R(cytb)/V135S(ISP),20 in which D
DOI: 10.1021/acs.biochem.5b00658 Biochemistry XXXX, XXX, XXX−XXX
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Table 1. Effect of UspA on the Electron Transfer and Superoxide Generation Activities of the Wild Type and Mutant bc1 Complexesa electron transfer activity % cytochrome bc1 complexes from various mutants wild type S287R(cyt b)/V135S(ISP) c1-14Gly-IV-6His ΔIV H198N H111N
100.0 122.5 139.3 33.9 10.7 7.2
± ± ± ± ± ±
superoxide generation activity %
+ UspA 1.7b 1.8 5.4 1.6 1.5 0.7
140.9 148.7 190.7 30.5 13.7 7.4
± ± ± ± ± ±
10.1 4.3 4.8 0.7 0.3 2.0
100.0 79.7 36.1 135.8 144.3 160.5
± ± ± ± ± ±
+ UspA 5.7c 4.1 3.3 5.7 6.9 6.5
75.1 49.8 22.5 72.9 102.5 108.2
± ± ± ± ± ±
4.5 3.7 2.8 4.1 5.4 4.9
a
The electron transfer and superoxide production activities in the wild-type and mutant complexes were determined in the presence or absence of an equal amount of UspA. The activity assay and superoxide production determination were as described in the Materials and Methods. N = 3, means ± SE. Additional discussion about the activity is in the Supporting Information. b100% refers to the activity values obtained with the wild-type complex, 2.5 μmol c red/min/nmol cyt b in the absence of UspA. c100% refers to the superoxide generation values obtained with the wild-type complex, 0.081 V in the absence of UspA.
Although it has been clearly demonstrated that UspA decreases superoxide production in Rsbc1 complex, it is unclear whether the observed decrease results from UspA scavenging the produced superoxide or blocking the superoxide production in Rsbc1. To answer this question, UspA scavenging activity was examined with superoxide generated by xanthine oxidase system. Since addition of UspA does not decrease the superoxide generated by xanthine oxidase (see Figure 4), the observed decrease in superoxide production must result from UspA blocking the superoxide production by Rsbc1.
Figure 4. Effect of UspA on superoxide generated by xanthine oxidase. The superoxide production by xanthine oxidase was the same as that described for the superoxide generation by the Rsbc1 complex in the Experimental Procedures. The level of superoxide produced by xanthine oxidase was adjusted to about the same level as that produced by Rsbc1. The concentration of xanthine oxidase is 0.9 μM. The black line represents superoxide production by xanthine oxidase plus UspA, and the gray line represents the superoxide production by xanthine oxidase only.
Figure 3. Effect of UspA on superoxide generation by the Rsbc1 complexes. Various amounts of UspA were added to a given amount of wild-type (A) and antimycin-treated (B) Rsbc1 complexes to give the molar ratios of Rsbc1 complex to UspA of 0, 1, 2, 3, and 4 as shown. Each treatment was incubated at room temperature for 5 min before superoxide production was measured.
How does UspA increase the electron transfer activity and block superoxide production activity by Rsbc1 under aerobic conditions? To answer this question, we first need to know whether or not UspA enhances the Rsbc1 activity under anaerobic conditions. As shown in Figure 5, no change in Rsbc1 activity was observed upon addition of UspA under anaerobic conditions. Thus, UspA increases Rsbc1 activity because it blocks electron transfer from the Rsbc1 complex to O2 to produce superoxide during the catalytic cycle.
inhibited Rsbc1 complex. When the concentration of UspA in the incubation mixture increased, superoxide production by Rsbc1 complex decreased. Maximum decrease in superoxide production (190 mV to 45 mV) was observed when 4 fold of UspA was present in the system. E
DOI: 10.1021/acs.biochem.5b00658 Biochemistry XXXX, XXX, XXX−XXX
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Figure 6. SDS-PAGE and Western blotting of sulfo-SBED cross-linked UspA and Rsbc1 complex. The experimental conditions for crosslinking UspA and Rsbc1 using sulfo-SBED were identifical as described in Experimental Procedures. The photolyzed sample was treated with ß-ME and subjected to SDS-PAGE and Western blotting. The biotin tags were probed with streptavidin-horseradish peroxidase conjugate. It shows that cytochrome c1 and UspA bind with biotin, which originally only stays on UspA. The stars represent contamination from the protein purification, which does not belong to either Rsbc1 or UspA.
Figure 5. Effect of UspA on the activity of Rsbc1 complex under aerobic and anaerobic conditions. The Rsbc1 complexes were diluted with the buffer containing 20 mM Tris-HCl, pH 8.0 at 4 °C, and 0.01% DM to a final concentration of cytochrome b of 1 μM. UspA was added to the Rsbc1 complex with a 1:1 molar ratio. The anaerobic conditions were achieved by a procedure involving repeated evaculation and flushing with argon gas. The activity of Rsbc1 complex only was used as 100%. N = 3, means ± SE.
Although the increase in activity of the Rsbc1 complex upon addition of UspA can be explained satisfactorily because of UspA’s prevention of electron leakage to O2, it is difficult to understand why the activity of the Rsbc1 complex under anaerobic conditions is lower than that under aerobic conditions. Under anaerobic conditions, there should be no electron leakage to O2, and therefore activity of the Rsbc1 complex would be expected to be higher but it is not. On the basis of the reduction kinetics of cytochromes b, a new electron transfer mechanism has recently been identfied24 for the Rsbc1 complex. Oxygen enhances the reduction rate of heme bL but shows no effect on the reduction rate of heme bH, suggesting that oxygen serves as an electron acceptor during bifurcated oxidation of ubiquinol. Identification of the Subunit of Rsbc1 that Interacts with UspA. To identify the subunit(s) in the Rsbc1 complex that interacts with UspA, a biotin-labeled transfer cross-linker, sulfo-SBED, was used as described under Experimental Procedures. UspA was first labeled with sulfo-SBED. SulfoSBED is a heterobifunctional chemical cross-linker which allows one to sequentially cross-link interacting proteins and transfer the biotin affinity tag from one protein to another. The sulfo-SBED labeled UspA was then photolyzed alone or in the presence of the Rsbc1 complex. The photolyzed samples were treated with ß-mercaptoethanol and then subjected to SDSPAGE and Western blot analysis (see Figure 6). The streptavidin horseradish peroxide conjugate was used to probe proteins having a transferred biotin tag. Five protein bands with apparent molecular masses of 43, 31, 30, 23, and 15 kDa were shown in the SDS-PAGE. Three protein bands with molecular masses of 40, 31, and 30 kDa were tagged with biotin and shown in the Western blot. Since UspA functions as a dimer, the detection of the transferred biotin tag in UspA itself was expected in the Western blotting as the 30-kDa band. The band with an apparent molecular mass of 31 kDa in the Western blotting had the same molecular masses as cyt c1. The bands of cytochrome b, ISP, and subunit IV, which showed in this SDS-PAGE, were not found in the Western blot (see Figure 6), which were not transferred with biotin from UspA.
The weak band with molecular mass of 40-kDa in the Western blotting did not show in the SDS-PAGE of UspA and Rsbc1, which means it does not belong to those two proteins, and it is considered as a nonspecific band of contamination from the protein purification. Only cyt c1 was shown both in the SDSPAGE and in the Western blot, indicating that the cytochrome c1 subunit transferred biotin from UspA and it is the site that interacts with UspA. Since the presence of subunit IV is required for UspA to exert activity enhancement of Rsbc1, subunit IV is most likely also involved in the interaction between UspA and Rsbc1. However, subunit IV might not have the direct interaction with UspA, but contributes to help the cytochrome c1 keep the proper structure and hold the binding site for UspA. Without subunit IV, the conformational change of Rsbc1 will lead the cytochrome c1 to lose the binding site for UspA; thus no enhancement of ΔIV Rsbc1 activity can be detected when UspA is added to the system.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.5b00658. Comments about the turnover number of our preparation from R. sphaeroides (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address #
Department of Environmental Health, Harvard T. H. Chan School of Public Health, Harvard University, Boston, MA 02115, USA. F
DOI: 10.1021/acs.biochem.5b00658 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry Funding
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This work was supported by Grant GM30721 from the National Institute of Health and by the Agricultural Experiment Station (Project 1819), Oklahoma State University. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS bc1, cytochrome bc1 complex; mbc1, mitochondrial bc1 complex; Rsbc1, bc1 from Rhodobacter sphaeroides; Q0C10BrH2, 2,3dimethoxy-5-methyl-6-(10-bromodecyl)1,4-benzoquinol; DM, N-dodecyl-ß-D-maltopyranoside; OG, N-octyl-ß-D-glucopyranoside; sulfo-SBED, sulfosuccinimidyl-2-[6-(biotinamido)-2-(pazidobenzamido) hexanoamido]ethyl-1,3-dithiopropionate); DEPC, diethylpyrocarbonate; TCEP, tris(2-carboxyethyl)phosphine; IPTG, isopropyl β-D-1-thiogalactopyranoside; MMLV RT, Moloney murine leukemia virus reverse transcriptase; dNTP, deoxy-ribonucleoside triphosphate; AA, antimycin A; PEG, polyethylene glycol
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DOI: 10.1021/acs.biochem.5b00658 Biochemistry XXXX, XXX, XXX−XXX
