A Spectrophotometric, Coupled Enzyme Assay to Measure the Activity of Succi‐ Nate Dehydrogenase

Accepted Manuscript A spectrophotometric, coupled enzyme assay to measure the activity of succi‐ nate dehydrogenase Andrew J.Y. Jones, Judy Hirst PII: S0003-2697(13)00336-9 DOI: http://dx.doi.org/10.1016/j.ab.2013.07.018 Reference: YABIO 11421 To appear in: Analytical Biochemistry Received Date: 4 June 2013 Revised Date: 9 July 2013 Accepted Date: 12 July 2013 Please cite this article as: A.J.Y. Jones, J. Hirst, A spectrophotometric, coupled enzyme assay to measure the activity of succinate dehydrogenase, Analytical Biochemistry (2013), doi: http://dx.doi.org/10.1016/j.ab.2013.07.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A spectrophotometric, coupled enzyme assay to measure the activity of succinate dehydrogenase† Andrew J. Y. Jones and Judy Hirst* The Medical Research Council Mitochondrial Biology Unit, Wellcome Trust / MRC Building, Hills Road, Cambridge, CB2 0XY, U. K. †This research was funded by The Medical Research Council. *Author to whom correspondence should be addressed: Medical Research Council Mitochondrial Biology Unit, Wellcome Trust / MRC Building, Hills Road, Cambridge, CB2 0XY, U. K. Tel : +44 1223 252810, Fax : +44 1223 252815, E-mail : [email protected] Short title: Coupled enzyme assay for succinate dehydrogenase Subject category: Enzymatic assays and analyses 1 Abstract Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate, and oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and NADP+ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (such as 2,6-dichlorophenolindophenol) our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions. Keywords: Coupled assay Fumarate hydratase Oxaloacetate decarboxylating malic enzyme Succinate:ubiquinone oxidoreductase 2 Introductory statement In mammalian mitochondria, succinate:ubiquinone oxidoreductase (succinate dehydrogenase, complex II, EC 1.3.5.1) catalyzes the oxidation of succinate to fumarate in the matrix, and the reduction of ubiquinone-10 in the inner membrane (1, 2). Thus, it links the tricarboxylic acid cycle to the respiratory electron transport chain. Complex II comprises four subunits (1-3). In the hydrophilic domain, the largest subunit houses a covalently-bound flavin adenine dinucleotide (FAD) cofactor and the succinate-binding site, and a smaller subunit contains three iron-sulfur clusters that transfer electrons from the flavin to ubiquinone. The binding site for the ubiquinone substrate is located close to a heme cofactor, in the membrane- associated domain that is composed of two hydrophobic subunits. Homologues of mammalian complex II are widely conserved in aerobic organisms, in both eukaryotes and prokaryotes. Some prokaryotes use fumarate as a terminal electron acceptor in anaerobic respiration, by using enzymes that are closely-related to complex II to catalyze the reverse reaction, reduction of fumarate to succinate (4). Some parasites, such as Ascaris suum, switch between succinate oxidation and fumarate reduction during different life stages, and the same switch has been proposed to occur in mammalian cells during hypoxia (5). Mutations in complex II have been identified as a cause of Leigh syndrome, a neurodegenerative disease (6), and complex II has recently been identified as a significant source of mitochondrial reactive oxygen species production (7). Complex II mutations have been linked also to hereditary paragangliomas, tumors in the head and neck (8), and there is currently significant interest in how mitochondrial succinate and fumarate levels are linked to the stabilization of HIF-1 and thus to cell proliferation (9). Due to its central role in metabolism and medicine, measurements of complex II activity have long been used to assess the activity of both the enzyme itself and of the mitochondrial electron transport chain as a whole. However, there is no convenient and 3 reliable method for testing complex II activity. Clark oxygen electrodes are often used (10) (see for example (7, 11)) but they are expensive in their sample requirements, and as they measure succinate:O2 oxidoreduction they can only assess the combined activity of respiratory complexes II, III and IV. Alternative real-time assays of complex II rely on artificial electron acceptors which change color upon reduction and can therefore be monitored spectrophotometrically. 2,6-dichlorophenolindophenol (DCPIP)1 and 2-(4-iodo- phenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) are reduced directly by complex II, and also by the quinol product of complex II catalysis (see, for example (12-14)). DCPIP and other tetrazolium salts (which do not react efficiently with complex II directly) have also been used as secondary electron acceptors, with phenazine methosulfate (PMS) to mediate electron transfer from complex II (see, for example, (12, 14, 15)). However, all of these assays lack specificity. When either the electron acceptor or PMS react with the enzyme directly they fail to focus on the complete catalytic cycle of succinate:ubiquinone oxidoreduction, the reactivity is not specific to complex II, and O2 interferes with the results (16). On the other hand, the reaction with ubiquinol is inefficient, electrons are not scavenged stoichiometrically when the respiratory chain is turning over, and the measured rate is always a combination from all possible pathways. Here, we present a convenient, stoichiometric and precise coupled enzyme assay for succinate oxidation (see Scheme 1). Our assay uses two enzymes: fumarate hydratase (FumC, EC 4.2.1.2) converts the fumarate product of succinate oxidation to malate (17), and then oxaloacetate decarboxylating malic dehydrogenase (MaeB, EC 1.1.1.40) couples the conversion of malate to pyruvate to the reduction of NADP+ to NADPH (18); the NADPH is detected spectrophotometrically. Because our assay quantifies succinate oxidation directly it is suitable for measuring succinate:ubiquinone oxidoreduction by isolated complex II, as well as succinate consumption in membrane and tissue preparations. 4 Materials and Methods Preparation of FumC and MaeB Fumarate hydratase (FumC) and oxaloacetate decarboxylating malic dehydrogenase from Escherichia coli (MaeB) were over-expressed in E. coli. Plasmid constructs encoding the fumC and maeB genes were supplied by Professor Todd Weaver (University of Wisconsin, La Crosse) (17) and Professor María Drincovich (National University of Rosario, Argentina) (18), respectively. The same protocol, adapted from refs. (17) and (18), was used for expression and purification of both proteins unless otherwise stated. BL21(DE3) E. coli cells (New England Biolabs) were transformed with the maeB or fumC plasmid. Cells were grown -1 at 32 °C in LB media supplemented with 50 µg mL ampicillin until A600 reached ~0.6, then protein expression was induced using isopropyl -D-1-thiogalactopyranoside (IPTG, 1 mM for FumC and 0.1 mM for MaeB) for 18 hours at 20 °C. Cells were harvested by centrifugation (10 mins., 3500 × g), resuspended in buffer A (50 mM potassium phosphate, 300 mM NaCl, pH 7.8 at 4 °C) for FumC or buffer B (20 mM Tris-Cl, 100 mM NaCl, 25 mM imidazole and 10 % w/v glycerol, pH 7.4 at 4 °C) for MaeB, then lyzed in one passage through a Constant Systems Ltd. model B 2.2 kW Z cell disruptor at 30 PSI. The lysates were centrifuged (45 mins., 150,000 × g), the pellets discarded, and the supernatants filtered (0.45 µm) before being loaded onto a pre-equilibrated 25 mL Ni-sepharose 6 fast flow column (GE Healthcare). The column was washed using buffer A + 60 mM imidazole for FumC or buffer B for MaeB, then the required proteins eluted in buffer A + 400 mM imidazole for FumC or buffer B + 300 mM imidazole for MaeB. Fractions were identified by activity assays, pooled and concentrated (Amicon Ultra-15 50 kDa centrifugal filter units), then dialyzed overnight in buffer C (10 mM Tris-SO4, 5 mM EDTA, 1 mM dithiothreitol, pH 7 at 4 °C) for FumC or buffer D (60 mM Tris-SO4, 20 mM β-mercaptoethanol, 25 mM imidazole and 10 % w/v 5 glycerol, pH 7.4 at 4 °C) for MaeB. Typical yields were 36 and 60 mg per liter of culture for FumC and MaeB, respectively. Preparation of complex II-containing samples Submitochondrial particles (SMPs) were prepared from bovine heart mitochondria as described previously (11). Complex II was solubilized from bovine heart mitochondrial -1 membranes (~10 mg protein mL in 10 mM Tris-SO4 pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.005% PMSF) by the addition of 0.5% n-dodecyl-β-D-maltopyranoside (DDM); the sample was stirred on ice for 30 mins., then centrifuged (30 mins., 48000 x g) and the supernatant collected.

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