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The Role of Phospholipids of Subunit III in the Regulation of Structural Rearrangements in Cytochrome c Oxidase of Rhodobacter sphaeroides Khadijeh S Alnajjar, Teresa L Cvetkov, and Lawrence John Prochaska Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi5013657 • Publication Date (Web): 05 Jan 2015 Downloaded from http://pubs.acs.org on January 12, 2015
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Biochemistry
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The Role of Phospholipids of Subunit III in the Regulation of Structural Rearrangements 5
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in Cytochrome c Oxidase of Rhodobacter sphaeroides. 6 7
Khadijeh S. Alnajjar#^, Teresa Cvetkov*, Lawrence Prochaska#** 10
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#
1
Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine at Wright
12
State University, Dayton, OH 13 14 ^
15 17
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Current address: Department of Therapeutic Radiology, School of Medicine at Yale University,
New Haven, CT 19
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*
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Current address: Max F Perutz Laboratories, Structural and Computational Biology, Vienna,
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Austria. 24
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KEYWORDS: Cytochrome c oxidase, Subunit III, Delipidated enzyme, Cardiolipin, V-Shaped 25 27
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Cleft, Förster Resonance Energy Transfer. 28 29
** CORRESPONDING AUTHOR 32
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Lawrence Prochaska 34
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937-775-2551 35 37
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[email protected] 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment
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ABSTRACT 5 7
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Subunit III of cytochrome c oxidase possesses structural domains which contain 9
8
conserved phospholipid binding sites. Mutations within these domains induce a loss of 10 1
phospholipid binding, coinciding with decreased electron transfer activity. Functional and 12 14
13
structural roles for phospholipids in the enzyme from Rhodobacter sphaeroides have been 16
15
investigated. Upon the removal of intrinsic lipids using phospholipase A2, electron transfer 17 18
activity was decreased 30-50 %. Moreover, the delipidated enzyme exhibited turnover-induced, 19 21
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suicide inactivation, which was reversed by the addition of exogenous lipids, most specifically 23
2
by cardiolipin. Cardiolipin exhibited two sites of interaction with the delipidated enzyme, a high 24 25
(Km = 0.14 μM) and low affinity (Km = 26 μM) sites. Subunit I of the delipidated enzyme 26 28
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exhibited a faster digestion rate when treated with α-chymotrypsin compared to wild-type 30
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enzyme, suggesting that lipid removal induces a conformational change to expose the digestion 31 3
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sites further. Upon reacting subunit III of the enzyme with a fluorophore (AEDANS), 35
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fluorescence anisotropy showed an increased rotational rate of the fluorophore in the absence of 37
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lipids, indicating increased flexibility of subunit III within the enzyme’s tertiary structure. 38 40
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Additionally, Förster resonance energy transfer between AEDANS and a fluorescently labeled 42
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cardiolipin revealed that cardiolipin binds in the v-shaped cleft of subunit III in the delipidated 43 4
enzyme and that it moves closer to the active site in subunit I upon a change in the redox state of 45 47
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the enzyme. In conclusion, these results show that the phospholipids regulate events occurring 49
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during electron transfer activity by maintaining the structural integrity of the enzyme at the 50 52
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active site. 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment
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Cytochrome c oxidase (COX) is a component of the electron transport chain located in 5 6
the mitochondrial inner membrane, which catalyzes the reduction of molecular oxygen to 9
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produce two water molecules while pumping one proton per electron transferred 1. The catalytic 1
10
core of COX is composed of three subunits that are highly conserved across species; they contain 12 13
the redox centers and are required for efficient activity 2. Subunit I (SUI) of COX contains heme 16
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a and the binuclear center, composed of heme a3 and CuB. Additionally, SUI contains two proton 18
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translocation pathways, the K- and D- channels, named after essential amino acids within the 19 20
channels, K362 and D132, respectively (Rhodobacter sphaeroides numbering) 3. Two of the 23
2
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substrate protons (used to reduce oxygen) are transferred through the K-channel, while the other 25
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two substrate protons, in addition to all pumped protons, are transferred through the D-channel 3. 26 28
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Subunit II (SUII) contains the cytochrome c binding domain and a diatomic-copper center (CuA). 30
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Subunit III (SUIII) is highly hydrophobic and does not contain metal centers. Several studies 31 32
suggested a possible role of this subunit in maintaining the structural integrity and rapid turnover 35
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rate through regulation of proton uptake, oxygen delivery and proton exit 4-9; however, the exact 37
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function has yet to be determined. 38 39
SUIII is located adjacent to SUI in the three dimensional structure of COX 3. It contains 42
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seven transmembrane α-helices that are arranged to form a v-shaped cleft with helices 1 and 2 as 4
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one bundle and helices 3-7 as the second bundle. The three-dimensional structure of SUIII from 45 47
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Rhodobacter 49
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sphaeroides
(RBS)
contains
six
bound
phospholipids,
identified
as
phosphatidylethanolamine (PE), which interact with the enzyme at different locations 3. SUIII 50 51
contains conserved phospholipid binding sites within the cleft, where two PE are intercalated 52 54
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within helices 2, 3 and 6 of SUIII 3. The other four phospholipids are positioned at the interface 56
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between SUI, SUIII and SUIV. The phosphate head groups of the phospholipids in the v-cleft of 57 58 59 60 ACS Paragon Plus Environment
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SUIII form electrostatic interactions with R137 (SUI) and R226 (SUIII), and the fatty acid chains 5
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form Van der Waals interactions with W58 and W59 as well as with other conserved residues in 7
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SUIII 10. 9
8 10
These lipids have been shown to be important in catalytic activity 12
1
10-12
. The removal of
the phospholipids weakens the interactions between SUI and SUIII, leading to the loss of SUIII 13 14 10
17
16
15
. Additionally, the enzyme undergoes turnover-induced, suicide inactivation in the absence of
the lipids, which leads to an alteration in the integrity of the active site and the ultimate loss of 19
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CuB. However, proton uptake rates through the D- and K- channels in SUI are unaffected by the 20 2
21
removal of those lipids 24
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10
. This suggests that SUIII contributes to efficient COX activity via
long-range interactions, which also stabilize the structure of the enzyme. Thus, it was proposed 25 26
that these lipids control the dynamic motion of the enzyme near the active site and that their 27 29
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absence causes increased flexibility at the active site, which consequently results in suicide 31
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inactivation 13. 32 3
Cardiolipin (CL) has been shown to be important for the activity of COX isolated from 36
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bovine heart mitochondria 12. The crystal structure of bovine heart COX shows two tightly bound 38
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CL surrounding the catalytic core 39
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. Upon the removal of these tightly bound CLs in bovine
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COX, steady-state activity decreases and some accessory subunits dissociate 12. The addition of 43
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CL to delipidated COX induced a recovery of activity to WT COX, which was not observed with 45
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phosphatidylcholine (PC) or PE 12. It has also been shown that the removal of tightly bound CL 46 48
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results in the loss of the low affinity site of cytochrome c interaction with COX 50
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11
. It was
proposed that the negatively charged CL aids in the docking of cytochrome c to COX by forming 52
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additional electrostatic interactions, which govern the interaction between the binding partners 15. 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment
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In support of this hypothesis, CL has been shown to be important in the activity of mitochondrial 5
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cytochrome bc1, 16, 17 18. 6 7
To test the role of phospholipids in the structural regulation of RBS COX, phospholipase 10
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A2 was used to remove the lipids. Results show that the delipidated COX exhibits biphasic 12
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kinetics in the presence of CL, and that the low affinity cytochrome c site for interaction is lost in 13 14
the absence of phospholipids. Additionally, Förster resonance energy transfer (FRET) studies 17
16
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between fluorescently labeled SUIII and a fluorescently labeled CL show that the phospholipids 19
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modulate the dynamic motion of COX during redox events. 20 2
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MATERIALS AND METHODS 24
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Expression and Purification of Cytochrome c Oxidase: Rhodobacter sphaeroides cells were 25 26
grown as previously reported in Zhen et al. 27
19
. WT COX was purified from the n-dodecyl β-D-
29
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maltoside (DM) solubilized membranes using affinity chromatography on a Ni+2-NTA column. 31
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The enzyme was eluted from the column with 100 mM histidine, washed, and concentrated by 32 3
ultrafiltration using 10 mM TrisCl, pH 8.0, 40 mM KCl, 0.1% DM using Millipore YM-100 36
35
34
filters. Concentration was determined spectrophotometrically (reduced-oxidized Δε606-630= 28.9 38
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mM-1cm-1) 20. SUIII depleted COX (I-II oxidase) was prepared by incubation in 12% Triton X39 40
100 for 30 minutes at 4 oC. The mixture was loaded onto a Ni-NTA column, washed with 10 mM 43
42
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TrisCl, pH 8.0, 40 mM KCl, 0.1% DM, and eluted with 100 mM histidine 21. 4 45
Removal of Phospholipids: WT COX was incubated with equimolar amounts of phospholipase 46 48
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A2 (PLA2) (from honey bee venom provided from Sigma-Aldrich, 600-2400 units/mg protein) in 50
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20 mM MOPS, pH 7.2, 10 mM CaCl2, 10 % glycerol, in the presence of 1 mg DM per mg COX. 51 53
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The reaction continued for three hours at 4 °C and was quenched with 50 mM EDTA
22
. The
5
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delipidated enzyme was diluted with 5 mM potassium phosphate, pH 8.0, 0.1 % DM, loaded 56 57 58 59 60 ACS Paragon Plus Environment
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onto a cytochrome c affinity column, eluted with 5 mM potassium phosphate, pH 8.0, 0.1 % DM, 5
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50 mM NaCl, and concentrated with Millipore YM-100 filters 23. 6 8
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Measurement of Phospholipid Content: Purified COX samples were incubated with 10 mM 10
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EDTA for 15 minutes and then washed three times in Millipore YM-100 filters with 10 mM 1 12
TrisCl, pH 7.4, 40 mM KCl prepared in metal-free water. Sixty-five µg of COX was diluted with 13 15
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three mL metal-free water. Standards of iron, copper and phosphorous were prepared at 17
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concentrations ranging from 0 to 75 µg/L. Samples were analyzed using an Elan 9000 18 19
inductively coupled plasma-mass spectrometer (ICP-MS) and the concentrations were 20 2
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intrapolated using a standard curve. Phosphorous was used to measure the phospholipid content 24
23
in the sample. Iron and copper from COX preparations were used as internal standards and data 25 27
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were normalized to three moles of copper per mol COX. 29
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Limited Proteolysis: COX was incubated with TLCK-treated α-chymotrypsin (Worthington) at 31
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a ratio of 10 COX: 1 chymotrypsin (w/w) in 20 mM TrisCl, pH 8.0 at 25 °C for up to 5 h and 32 34
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stopped using 1 mM PMSF. The digested enzyme was incubated with 2 % SDS at 37 °C for 30 36
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minutes and then run on a SDS-urea polyacrylamide gel 37
24
. Gels were stained with Coomassie
38
Blue and viewed using a Fuji LAS-3000 Imager. The staining intensity of SUI was measured 39 41
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using Multi Gauge and plotted as a function of digestion time. 43
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Recovery of Oxygen Reduction Activity: Oxygen reduction activity was measured 4 45
polarographically in 50 mM potassium phosphate, pH 7.4, 0.1 % DM in the presence of 17 mM 46 48
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ascorbic acid, 0.6 mM TMPD and 20 µM cytochrome c at 25 °C. Lipids were solubilized in 50
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assay buffer by sonication to clarity at 4 °C. COX (2 µM) was incubated with each lipid ligand 51 53
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for at least 15 minutes prior to assay. 54 5 56 57 58 59 60 ACS Paragon Plus Environment
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Steady-State Cytochrome c Kinetics: The cytochrome c concentration dependence of COX 5
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electron transfer activity was measured polarographically in 25 mM Tris-acetate, pH 7.8, 0.1 % 6 7
DM, in the presence of 17 mM ascorbic acid and 0.6 mM TMPD at 25 °C. 25 Cytochrome c was 10
9
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varied in concentration from 0.1-10 µM. The enzyme was pre-incubated with 500 μM CL where 1 12
indicated. 13 15
14
Labeling with IAEDANS: COX was incubated with IAEDANS at a molar ratio of 10 17
16
IAEDANS: 1 COX in 20 mM HEPES, pH 8.0, 0.1 % DM at 25 °C and in the dark for 1 h 26. The 18 19
reaction was quenched with 100 fold molar excess of dithiothreitol and washed a minimum of 20 2
21
three times with 20 mM HEPES, pH 8.0 using Millipore YM-100 filters. Potassium ferricyanide 24
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was used to oxidize the enzyme. The binding stoichiometry of AEDANS to COX was 26
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determined using the absorbance at 336 nm (ε336 = 5.7 mM-1 for AEDANS) 29
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27
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in a Hewlett
Packard 2451 diode array spectrophotometer and corrected for the absorbance of oxidized COX 31
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at 336 nm (ε336 = 44 mM-1 for COX). SDS-PAGE was used to test the specificity of labeling to 32 34
3
SUIII and AEDANS fluorescence was monitored (excitation at 312 nm) using a Fuji LAS-3000 36
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Imager 24. Gels were subsequently stained with Coomassie Blue. 37 38
Förster Resonance Energy Transfer (FRET): TopFluor labeled cardiolipin (TFCL) (1,1',2,2'39 41
40
tetraoleoyl cardiolipin [4-(dipyrrometheneboron difluoride) butanoyl] (ammonium salt)) (Avanti 43
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Polar Lipids) was solubilized in 50 mM potassium phosphate, pH 7.4, 50 µM DM. FRET from 45
4
AEDANS-labeled COX (donor, λex 336 nm, λem 470 nm) to TFCL (acceptor, λex 490 nm, λem 505 46 48
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nm) was measured using an ISS PC1 spectrofluorimeter with 1 mm slits. One µM TFCL was 50
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added to 0.5 µM AEDANS-labeled COX in 50 mM potassium phosphate, pH 7.4, 50 µM DM, 51 53
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and energy transfer was monitored as a function of time. FRET was measured with the oxidized 5
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enzyme and in the presence of 0-40 mM ascorbic acid. For distance measurements, the refractive 56 57 58 59 60 ACS Paragon Plus Environment
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index was assumed to be 1.43 in a solution of 0.1 % DM 5
4
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donor in the absence of the acceptor is reported as 0.94 6
, fluorescence quantum yield of the
29
. Additionally, the dipole angular
7
orientation factor was assumed to be at 2/3 for dynamic motion of the donor and the acceptor 30. 10
9
8
These assumptions were used to calculate the Förster distance to be 53.1 ± 0.4 Å. The efficiency 1 12
of energy transfer was calculated using the quenching of AEDANS emission by TFCL and 13 14
corrected for the inner filter effect resulting from the absorbance of COX and ascorbic acid 31. 17
16
15
For all the reported results, experiments were performed a minimum of three times and 18 19
the average with standard deviations are reported. 20 2
21
RESULTS 24
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The removal of endogenous lipids from RBS COX results in reduced steady-state activity 25 27
26
and in suicide inactivation
10, 11
. These events indicate that lipids play an important role in the
29
28
regulation of structural flexibility at the active site of SUI, which in turn modulates steady-state 31
30
activity. Thus, this study is aimed to clarify the role of phospholipids in the structure and 32 34
3
function of COX. 36
35
Removal of the Phospholipids from RBS COX 37 38
WT COX was treated with molar stoichiometric amounts of PLA2 in the presence of DM 39 41
40
to remove the phospholipids embedded in the enzyme without inducing major perturbations in 43
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SUI-SUIII interactions 4
12, 18, 21
. Loss of phospholipids by the treatment was confirmed by ICP-
45
MS as the ratio of phosphorous per mole COX (Table 1). Iron and copper (from COX) were used 46 48
47
as internal standards and phosphorous was quantified relative to three moles of copper. Table 1 50
49
shows that purified WT COX retained 5-6 moles of phosphorous as phospholipids per mole 51 53
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COX. Upon treatment with PLA2, the enzyme lost most of its phospholipid content (
