View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 221, number 2, 343-348 FEB 05097 September 1987 The interaction of ferredoxin-linked sulfite reductase with ferredoxin Masakazu Hirasawa, J. Milton Boyer, Kevin A. Gray, Danny J. Davis* and David B. Knaff Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-4260 and *Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA Received 22 June 1987; revised version received 21 July 1987 Spinach sulfite reductase has been shown to co-migrate during gel filtration chromatography at low ionic strength with spinach ferredoxin. No co-migration was observed at high ionic strength. These results indic- ate that the two proteins form a high-affinity, electrostatically stabilized complex, as had previously been demonstrated for three other ferredoxin-dependent, plant enzymes. Modification of 3-4 ferredoxin carboxyl groups had little detectable effect on the ferredoxin-sulfite reductase interaction. Sulfite-reductase; Ferredoxin; (Spinach) 1. INTRODUCTION synthase (EC 1.4.7.1). Chemical modification [12,13], cross-linking [10,13,14] and NMR studies A key step in sulfate assimilation by higher [ 151 have implicated carboxyl groups on ferredoxin plants [l] is the six-electron reduction of sulfite to as supplying the negative charges involved in form- sulfide, catalyzed by the enzyme ferredoxin : sulfite ing these complexes. As ferredoxin-dependent oxidoreductase (EC 1.8.7.1, hereafter referred to spinach sulfite reductase can be purified using a as sulfite reductase). Sulfite reductase, located in ferredoxin-Sepharose 4B affinity column [3], it ap- the chloroplast [2], has been purified to peared likely that sulfite reductase, like the other homogeneity from spinach leaves [3-51. The en- plant enzymes listed above, can form an elec- zyme, which contains one siroheme and one trostatic complex with ferredoxin. Evidence for [4Fe-4S] cluster as prosthetic groups [3-51, can such complex formation is presented below. utilize reduced methyl viologen as an electron donor [3,4] but the physiological electron donor is 2. MATERIALS AND METHODS reduced ferredoxin [3,4]. Considerable evidence exists [6-l l] that fer- Spinach ferredoxin (,4422nm:A277nm= 0.45) was redoxin forms electrostatically stabilized com- prepared according to Tagawa and Arnon [16]. plexes with a number of ferredoxin-dependent Ferredoxin was modified by treatment with glycine plant enzymes, including ferredoxin : NADP+ ox- ethyl ester in the presence of 1-ethyl-3-(3-dimethyl- idoreductase (EC 1.18.1.2, hereafter referred to as aminopropyl)carbodiimide (EDC) according to NADP+ reductase), ferredoxin : nitrite oxidoreduc- Vieira and Davis [12]. Sulfite reductase was tase (EC 1.7.7.1, hereafter referred to as nitrite purified by a modification of the procedure of reductase) and ferredoxin-dependent glutamate Aketagawa and Tamura [3]. An isolation buffer consisting of 100 mM Tris-HzS04 (pH 7.9, Correspondence address: D.B. Knaff, Department ot 200 mM NaCl, 2 mM Na2S03, 1 mM EDTA and Chemistry and Biochemistry, Texas Tech University, 1 mM phenylmethylsulfonyl fluoride replaced that Lubbock, TX 79409-4260, USA used previously. Acetone precipitation and anion- Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/87/$3._50 0 1987 Federation of European Biochemical Societies 343 Volume 221, number 2 FEB.5 LETTERS September 1987 exchange chromatography were carried out as in redoxin was omitted from the reaction mixture, [3] except that the O-50% acetone precipitate, rates of sulfite and enzyme-dependent oxidation of after re-dissolving in buffer, was concentrated by reduced methyl viologen approx. 15% of those ob- precipitation with 70% saturated ammonium tained with the complete reaction mixture were sulfate and dialyzed prior to chromatography on a observed. Whatman DE-52 DEAE cellulose column that had Absorbance spectra were obtained using an been equilibrated with 50 mM Tris-HzSO4 buffer Aminco DW-2a spectrophotometer and CD spec- (pH 7.5) containing 50 mM NaCl, 0.5 mM EDTA tra were obtained using a Jasco J-20 spec- and 20% (v/v) glycerol. Active fractions were tropolarimeter . Oxidation-reduction titrations pooled, concentrated by precipitation with 70% were performed electrochemically using the op- saturated ammonium sulfate and, after dialyses, tically transparent gold electrode/thin-layer cell applied to an Ultrogel AcA 34 gel filtration column system in [17]. Protein concentration was deter- that had been equilibrated with 50 mM Tris-HzS04 mined according to Bradford [ 181, using bovine buffer (pH 7.5) containing 200 mM NaCl, 0.5 mM serum albumin as a standard. Ferredoxin concen- EDTA and 20% glycerol. Active fractions were trations were determined using 6422”rn = 9.7 X pooled, concentrated by ammonium sulfate pre- lo3 M-’ *cm-’ [16]. Sulfite reductase concentra- cipitation, dialyzed against 10 mM potassium tion was calculated from protein concentration, phosphate buffer (pH 7.5) containing 140 g/l of assuming a molecular mass of 138 kDa [4]. ammonium sulfate, applied to a phenyl-Sepharose CL4B column equilibrated with the same buffer and eluted as described by Krueger and Siegel [4]. 3. RESULTS Fractions containing sulfite reductase with specific Since the purification protocol used to obtain activity > 2.0 U/mg protein (see below) were pool- the spinach sulfite reductase for the experiments to ed, concentrated by ammonium sulfate precipita- be described below differed somewhat from those tion, dialyzed against 10 mM potassium phosphate reported previously [3,4], it seemed appropriate to buffer (pH 7.7) containing 10% glycerol and ap- compare some of the preparation’s kinetic proper- plied to a ferredoxin-Sepharose 4B affinity column ties with literature values [3,4]. The enzyme obeyed [3] equilibrated with the same buffer. After Michaelis-Menten kinetics with respect to the con- washing the column with the equilibration buffer centration of the electron donor. K,,, values of 25 to remove undesired protein, sulfite reductase was and 6 PM were obtained for reduced ferredoxin eluted with 100 mM potassium phosphate buffer and reduced methyl viologen, respectively. When (pH 7.7). Fractions containing high specific activi- the enzyme was assayed (using reduced ferredoxin ty were pooled and concentrated by membrane as the electron donor) at varying sulfite concentra- filtration using an Amicon PM-10 membrane. The tions, a sigmoidal relationship between rate and final enzyme preparation had a ratio of sulfite concentration similar to that reported by A3s5,,,,,:A280,,,,, of 0.20 and a specific activity of Aketagawa and Tamura [3] was observed. Half- 5.3 U/mg protein with reduced ferredoxin as the maximal velocity was observed at 120pM sulfite electron donor. and a Hill coefficient [ 191 of 2.1 (calculated by fit- Sulfite reductase was assayed by monitoring the ting the kinetic data using a linear least-squares absorbance decrease at 604 nm due to the oxida- regression program - GRAPH, Version 1 from tion of reduced methyl viologen, using a modifica- Cricket Software - on a Macintosh Plus computer) tion of the procedure of Aketagawa and Tamura was calculated. The visible absorbance’ spectrum [3]. The reaction mixture (under an Nz atmosphere (not shown) and apparent molecular mass, deter- in a Thunberg cuvette) contained, in a total volume mined by gel filtration (see below), were essentially of 2.5 ml, 100,~mol Tris-H2SOd buffer (pH 8.0), identical to those reported in [3,4]. 3 mg bovine serum albumin, 40 nmol ferredoxin, The observation that spinach sulfite reductase 5 pmol Na2S03, 5 rmol Zn-reduced methyl can be purified using a ferredoxin-Sepharose 4B viologen and enzyme. 1 unit of activity was defined affinity column [3] strongly suggested that fer- as the amount of enzyme catalyzing the oxidation redoxin forms a complex with the enzyme. Addi- of 1 pmol reduced methyl viologen per min. If fer- tional evidence for complex formation comes from 344 Volume 221, number 2 FEBS LETTERS September 1987 I Ferredoxin 1.0- \ la- 0 1.7- \ 0 oIsultlt.Reductme Z ‘0 ; Sutttte Reducbise . Natlva Ferredoxin 1.6- Sutttte Reducta~ - Modltied Femdoxin t 10’ 105 Fig. 1. Gel filtration chromatography of ferredoxin plus Log Molecutar Weight sulfite reductase. Chromatography was performed on an Fig.2. Apparent i14~ values of ferredoxin-sulfite Ultrogel AcA 34 column (1 x 30 cm). (A) Elution in reductase complexes. Chromatography was performed 30 mM Tris-HCl buffer (pH 8.0). (A---A) Data for as described in fig.lA. M, standards used bovine carboxyl-modified ferredoxin plus sulfite reductase; (0): (M) data for unmodified, native ferredoxin plus serum albumin (Mr = 66000); hexokinase (Mr = sulfite reductase. (B) Elution in 30 mM Tris-HCl buffer 110000); alcohol dehydrogenase (Mr = 150000) and (pH 8.0) containing 200 mM NaCl. The data were bovine liver catalase (Mr = 240000). obtained from an experiment with sulfite reductase plus unmodified, native ferredoxin. the observation (not shown) that when sulfite apparent molecular mass for the ferredoxin-sulfite reductase was added to a ferredoxin-saturated reductase complex (fig.2) was 160 + 6 kDa (deter- DEAE-cellulose column, ferredoxin and sulfite mined according to Andrews [20]). No protein was reductase were co-eluted when 20 mM Tris-HCl detected in fractions corresponding to molecular buffer (pH 8.0) containing 80 mM NaCl was ap- masses near