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Human Indoleamine 2,3-Dioxygenase 1 is an Efficient Mammalian Nitrite Reductase Yean J Lim, Timothy C Foo, Amanda Yeung, Xiaofan Tu, Yuanqing Ma, Clare L. Hawkins, Paul K Witting, Guy N. L. Jameson, Andrew C. Terentis, and Shane R. Thomas Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01231 • Publication Date (Web): 26 Dec 2018 Downloaded from http://pubs.acs.org on January 3, 2019
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Biochemistry
Human Indoleamine 2,3-Dioxygenase 1 is an Efficient Mammalian Nitrite Reductase Yean J. Lim1, Timothy C. Foo3, Amanda W.S. Yeung1, Xiaofan Tu1, Yuanqing Ma2, Clare L. Hawkins6, Paul K. Witting4, Guy N. L. Jameson5, Andrew C. Terentis3, and Shane R. Thomas1,*
1Mechanism
of Disease and Translational Research, Dept. of Pathology or 2Single Molecule Science,
School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia; 3Department of Chemistry & Biochemistry, Florida Atlantic University, Florida, USA; 4Discipline of Pathology, Charles Perkins Centre, Faculty of Medicine and Health, University of Sydney, NSW, Australia; 5School of Chemistry, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC, Australia; 6Department of Biomedical Sciences, University of Copenhagen, Denmark.
KEYWORDS: heme dioxygenase, nitric oxide, hypoxia.
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ABSTRACT The heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of L-tryptophan oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders and cancer. Here we demonstrate that IDO1 is a mammalian nitrite reductase capable of chemically reducing nitrite to nitric oxide (NO) under hypoxia. UV-visible absorption and resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous-IDO1 protein (FeII-IDO) with nitrite under anaerobic conditions resulted in the time-dependent formation of a FeII-nitrosyl IDO1 species, which was inhibited by substrate Ltryptophan, dependent on the concentration of nitrite or IDO1 and independent of the concentration of the reductant, dithionite. The bimolecular rate constant for IDO1 nitrite reductase activity was determined as 5.4 M-1 s-1 (pH 7.4, 23C), which was comparable to that measured for myoglobin (3.6 M-1 s-1; pH 7.4, 23C), an efficient and biologically important mammalian heme-based nitrite reductase. IDO1 nitrite reductase activity was pH-dependent but differed with myoglobin in that it showed a reduced proton dependency at pH>7. Electron paramagnetic resonance studies measuring NO production showed that the conventional IDO1 dioxygenase reducing co-factors, ascorbate plus methylene blue, enhanced IDO1’s nitrite reductase activity and the time- and IDO1 concentration-dependent release of NO in a manner inhibited by L-tryptophan or the IDO inhibitor 1-methyl-L-tryptophan. These data identify IDO1 as an efficient mammalian nitrite reductase that is capable of generating NO under anaerobic conditions. IDO1’s nitrite reductase activity may have important implications for the enzyme’s biological actions when expressed within hypoxic tissues.
INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO1, UniProtKB P14902) is a 42-45 kDa, cytosolic heme enzyme that catalyzes the initial and rate limiting reaction of Ltryptophan (L-Trp) metabolism along the kynurenine pathway (1, 2). A large body of evidence indicates that IDO1 is an important immune regulatory enzyme capable of controlling T cell activation, phenotype and viability (1-3). In general, IDO1 inhibits T cell function resulting in the suppression of immune responses relevant to a variety of physiological and pathological conditions, including pregnancy, allergic and autoimmune disorders, inflammatory disease, infection and cancer (1, 2). While IDO1’s immune suppressive activity is reported to provide protection against animal models of inflammatory and autoimmune disease, IDO1 is increasingly recognized as a mechanism employed by tumor cells to evade immune surveillance and clearance (1, 4, 5). Accordingly, IDO1 pathway inhibitors, such as 1-methyl-tryptophan, are currently being tested in clinical trials as novel immunotherapeutic adjuvants in cancer patients (5). The discovery of IDO1’s role as a key immune regulatory enzyme and its clinical relevance has reinvigorated interest in understanding the fundamental biochemistry of the enzyme. IDO1 is a member of a distinct family of mammalian heme dioxygenases (1). Dioxygenase activation requires a one-electron (e−) reduction of IDO1’s active-site heme from the inactive ferric-iron (FeIII) to the active ferrousiron (FeII) state (Scheme 1) (6, 7). The FeII-IDO1 species binds O2 and L-Trp, forming the active ternary complex that initiates a complex set of redox reactions involving the formation of a ferryl (FeIV)-intermediate (8-10) and oxidative cleavage of the pyrrole ring of L-Trp to ultimately re-form the native FeIII-IDO1 and product Nformyl-kynurenine (Scheme 1), which decomposes into the more stable product kynurenine (6).
Scheme 1. Dioxygenase reaction of IDO1 While the superoxide anion radical (O2•-) was initially considered the physiological reductant for IDO1, more recent work suggests a role for cytochrome b5 reductase (11, 12). In vitro IDO1 dioxygenase assays commonly employ ascorbate and methylene blue as reducing cofactors; e− transfer to methylene blue from ascorbate yields reduced leuco-methylene blue, which mediates the FeIII- to FeII-heme redox cycling necessary for IDO1 activity (6, 7, 13). Under O2-deprived or hypoxic conditions characteristic of tissue ischemia, infection or within tumors, the dioxygenase activity of IDO1 is inhibited (14, 15). Similarly, the enzyme activity of nitric oxide synthase (NOS) enzymes is also impaired during hypoxia resulting in the inhibition of nitric oxide (NO) production (16). However, under reduced O2 tensions certain mammalian metalloproteins, including heme proteins, can exhibit a nitrite reductase activity that refers to their ability to catalyze the one-electron reduction of nitrite (NO2-) into NO (17-19). Mammalian proteins capable of catalyzing the reduction of NO2include the molybdenum proteins xanthine oxidase (20) or mitochondrial amidoxime reducing component (mARC) proteins (21) and several heme proteins including the endothelial isoform of NOS (eNOS) (22, 23), cytochrome c (24), and members of the globin family, including haemoglobin (25, 26), myoglobin (27, 28), cytoglobin (29, 30) and neuroglobin (31, 32). The nitrite reductase activity of heme proteins involves the reduction of NO2- by the FeII-heme to form NO and the FeIII-heme (Reaction I).
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Biochemistry
FeII + NO2- + H+ → FeIII + NO + OH- (Reaction I) NO +
FeII
→
FeII-NO
(10 µM) in the presence of phenyl-methane-sulfonylfluoride (PMSF; 500 µM) and hemin (3.5 µM). After 3 h incubation at 30°C in a shaking incubator the reaction was stopped with EDTA (1 mM) and the bacteria harvested by centrifugation (3500 g, 10 min, 4°C). Pellets were re-suspended in phosphate buffered saline (PBS, pH 7.4) containing 1 mM EDTA and 1 mM PMSF and re-isolated by centrifugation (27,200 g, 15 min, 4°C). These secondary pellets were re-suspended in ice-cold PBS containing complete EDTA-free protease inhibitor cocktail (Roche) and lysed by sonication (Branson Sonifer 250 with a 102C converter attachment and a flat tip attached to a half-inch horn). The lysate was centrifuged (27,200 g, 15 min, 4°C), the supernatant fraction collected and sequentially filtered through 45 and 22 µm membranes. The IDO1 protein was purified with a 5 mL His-Tag column (HisTrap HP, GE Life Sciences) pre-equilibrated in binding buffer (20 mM phosphate buffer with 20 mM imidazole) in a FPLC DuoFlow machine (BioRad) at 4°C. The column was washed with 10 column volumes of binding buffer at 5 mL/min and the protein eluted with 20 mM sodium phosphate buffer containing 150 mM imidazole at 3 mL/min. Heme-containing fractions were pooled and concentrated in a 10 kDa molecular weight cut-off spin column (Amicon Ultra 15, Millipore). Concentrated IDO1 was injected into a size-exclusion column (Superdex HiLoad 16/60, 200 pg, GE Life Sciences) and eluted in sodium phosphate buffer (20 mM) at 1 mL/min (4°C) in FPLC DuoFlow machine (BioRad). Fractions containing the IDO1 monomer were pooled and concentrated as above and the concentrate gel-filtered (PD-10, GE Life Sciences) into Tris-HCl (50 mM, pH 7.4). IDO1 concentration was determined by UV-visible absorption spectroscopy (ε404 = 159 mM-1 cm-1). Purified IDO1 protein exhibited a heme:protein absorbance ratio (A404:A280) of >1.7. For experiments, stock IDO1 protein solution was diluted in 50 mM potassium phosphate buffer (pH 7.4). For anaerobic reactions, stock IDO1 solution was gently purged over the solution surface with argon, while buffers and other reagents were degassed by purging argon through the solutions for >10 min. For heat-inactivated IDO1, the protein was incubated at 100°C for 2 min.
(Reaction II)
In this reaction, the activated FeII-heme catalyzes the cleavage of the NO bond of NO2-, with the resultant oxygen atom accepting a hydrogen ion (H+) to form OH(Reaction I). Therefore, the nitrite reductase activity of heme proteins is accelerated under acidic environments (28, 29, 31). In the absence of competing reaction targets and presence of an efficient heme-iron reductant, the NO generated in Reaction I can rapidly react with the excess FeII-heme to form a FeII-nitrosyl (FeII-NO) enzyme species (Reaction II). Considerable evidence supports that the nitrite reductase activity and resultant production of NO by these various enzymes contributes to the control of physiological responses such as hypoxic vasodilation (33), the control of cellular mitochondrial respiration (28) and maintenance of endothelial barrier integrity (34), as well as protection against various hypoxic pathologies including pulmonary hypertension and oxidative ischemia/reperfusion tissue injury (17, 27). Given their biological importance and protective actions, there is great interest in the discovery of new mammalian nitrite reductases. Employing recombinant human IDO1 protein combined with UV-Vis absorption, resonance Raman and electron paramagnetic resonance spectroscopic techniques we provide new data indicating that human IDO1 is a novel mammalian heme-based nitrite reductase capable of reducing NO2- to generate NO under anaerobic conditions. EXPERIMENTAL METHODS Materials. Chemicals and reagents were purchased commercially: NOC-9 (Santa Cruz Biotechnology) and N-Methyl-D-glucamine dithiocarbamate (Enzo Life Sciences). Unless otherwise stated, all other laboratory chemicals, reagents and proteins were purchased from Sigma Aldrich (Castle Hill, Australia) or Ajax Finechem (Australia). The concentrations of stocks of the following compounds were determined by UV-visible absorption spectroscopy using the relevant extinction coefficients: L-Trp, ε280 = 5690 M-1 cm-1; kynurenine, ε365 = 4530 M-1 cm-1; NOC-9, ε250 = 7800 M-1 cm-1, aqueous nitrite, ε354 = 22.93 M-1 cm-1.
UV-Visible Absorption Spectroscopy. Changes to the UV-visible absorption heme spectra of IDO1 or horse myoglobin (Mb; Sigma) (3–7 μM) were measured in PTFE-capped or septum-seal capped (Starna), 1-cm path-length quartz cuvettes using a Varian Cary 300 spectrophotometer (Agilent Technologies) with an attached water thermostat set to 23°C (room temperature). For nitrite reductase kinetic studies, anaerobic reactions were routinely monitored in 50 mM phosphate buffer at pH 7.4 or where relevant in different pH buffers (pH 6.4–8.0) in the absence or presence of dithionite (0.25–2.5 mM), L-Trp (0.5 or 1 mM) and NO2- (0.1–1 mM). All reaction buffers contained 0.1 M DETAPAC to chelate free metal ions and prevent redox
Recombinant Human IDO1. Recombinant human IDO1 protein was expressed in E. coli EC538 strain transformed with the pREP4 and pQE9-IDO1 plasmids and purified as previously described (35, 36), but with modifications. A bacterial starter culture was initiated in 20 mL of Luria-Bertani (LB) medium containing ampicillin (100 µg/mL) and kanamycin (50 µg/mL) and incubated overnight (< 15 h) in a shaking incubator at 30°C. Starter cultures were transferred into 1 L of LB medium and incubated at 30°C until an optical density at Abs600nm of 0.6 was achieved. IDO1 protein expression was induced with isopropyl-β-D-thiogalactopyranoside
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mM) and methylene blue (25 μM) co-factors plus the NO-reactive FeII(MGD)2 spin trap complex (0.5 mM) in the absence or presence of L-Trp, 1-methyl-Dtryptophan or 1-methyl-L-tryptophan (all 1 mM) under anaerobic conditions. The spin trap complex FeII(MGD)2 was freshly prepared by mixing N-Methyl-D-glucamine dithiocarbamate (10 mM) with ferrous sulfate heptahydrate (2 mM), both dissolved in MilliQ water, at a 1:1 (v:v) ratio.(40-43) The FeII(MGD)2 complex was then added to the reaction solution at a 1:1 volumetric ratio such that the final concentration of FeII in the complex was 0.5 mM. The reaction solution was incubated under anaerobic conditions for different times up to 30 min at 37°C, before being snap frozen at -80°C and stored for
