GREENE, R. C. (1970) in The Current Status of Liquid Scintilla- tion Counting. (BRANSOME,. E. D., JR., ed) p. 189, Grune and. Stratton,. New York. KOBAYASHI,.
THE JOURNAL OF Bmmorc~~ CHEMI~TBY Vol. 249, No. 5,Issue of March 10, pp. 1572-1586, 1974 P~inled
in U.S.A.
Reduced Nicotinamide Adenine Dinucleotide Sulfite Reductase of Enterobacteria III.
THE ESCHERICH1/l COLI SEQUENCE OF ELECTRON
HEMOFLAVOPROTEIN: FLOW*
CATALYTIC
Phosphate-
PARAMETERS
AND
THE
(Received for publication, August 10, 1973) LEWIS
M.
SIEGEL,$
PATRICIA
S.
DAVIS,
AND
From the Veterans Administration Hospital Medicine, Durham, North Carolina 27710
HENRY
KAMIN
and the Department
of Biochemistry,
Duke
University
School of
SUMMARY
produced,
* Thesestudies were supported in part by Research Grants AM-13460and AM-040663 from the National Institutes of Health, and GB-7905 from the National Science Foundation, Administration Project No. 7875-01. $ To whom inquiries should be addressed.
Veterans
and no sulfur compounds
of oxidation
state between
sulfite and sulfide were detected during sulfate assimilation in E. coli (2). hlager (3) partially purified the E. coli enzyme responsible
for the sulfite-dependent oxidation of NADPH, and was strongly inhibited by cyanide.
reported that the activity 1572
Downloaded from www.jbc.org by guest, on July 25, 2011
while permitting retention of FAD and heme. This treatment inhibits all pyridine nucleotide-dependent reactions of NADPH-sulfite reductase (EC 1.8.1.2) from Escherichia the enzyme except transhydrogenase and FMN reductase. co2i is a complex hemoflavoprotein, molecular weight 670,000, The methyl viologen-sulfite reductase is unaffected. The containing 4 FMN, 4 FAD, 20 to 21 atoms of iron, 14 to 15 development of fluorescence due to FMN release parallels labile sulfides, and 3 to 4 moleculesof a novel type of heme the development of the observed inhibitions. The FAD of per enzyme molecule. This heme has been identified as an the FMN-free enzyme is reducible by NADPH, but the heme octacarboxylic iron-tetrahydroporphyrin (MURPHY, M. J., is not. If exogenous FMN is added, the heme becomes SIEGEL, L. M., KAMIN, H., AND ROSENTHAL, D. (1973) J. reducible and all NADPH-dependent activities are restored. Bid. Chem. 248, 2801). The enzyme catalyzes the stoiWe have concluded that electron flow from NADPH to chiometric conversion of sulfite to sulfide at the expense of sulfite follows the minimum linear sequence: 3 NADPH. The K,,, values for sulfite and NADPH are NADPH + FAD + FMN ---f heme -+ sulfite both 4 to 5 pM. Reduced methyl or benzyl viologens can serve as electron donors for sulfite reduction, but NADH In this scheme,FAD serves as the “entry port” for electrons cannot. In addition to sulfite reduction, the enzyme catfrom NADPH. It can transfer electrons directly to FMN alyzes the NADPH-dependent reduction of a variety of (internal or external) or to pyridine nucleotides and their “diaphorase” acceptors (cytochrome c, ferricyanide, 2,6- analogues. The heme is required for electron transfer to dichlorophenolindophenol, menadione, FMN, FAD) as well sulfite (and nitrite and hydroxylamine). The FMN is reas NADPH oxidase, NADPH-3-acetylpyridine adenine di- quired for electron transfer from the reduced FAD to the nucleotide phosphate transhydrogenase, NADPH-nitrite and heme (and hence to acceptors dependent on the heme) or, -hydroxylamine reductase and reduced methyl viologenmore directly, to diaphorase-type acceptors and OZ. ReNADPf reductase activities. All NADPH-dependent activ- duced methyl viologen can donate electrons to both the ities examined were competitively inhibited by NADP+. FMN and heme. The patterns of inhibition by a variety of Agents which react with the heme prosthetic group, i.e. salts of the NADPH-cytochrome c and reduced methyl CO, cyanide, and arsenite, inhibit the reductions of sulfite, viologen-sulfite reductase reactions are consistent with the nitrite, and hydroxylamine (with either NADPH or reduced hypothesis that these two reactions involve independent methyl viologen as electron donor), while all other activities portions of the enzyme molecule. are unaffected. Cyanide and CO binding to and C-0 dissociation from the enzyme (determined spectrophotometrically) parallel the respective development and relief of inhibition of NADPH-sulfite reductase activity. Development of inhibition requires the presence of reductant (NADPH) as Ellis (1) has shown that the reduction of sulfite to sulfide, a well as inhibitor, in accord with the observation that CO, cyanide, or arsenite can react with reduced, but not oxidized 6-electron reduction on the pathway of cysteine biosynthesis, can be catalyzed by crude extracts of Escherichiacoli in a reenzyme. on NADPH as reductant. Such crude Treatment of enzyme with 1 PM p-chloromercuriphenyl- action dependent sulfonate causesthe dissociation of virtually all of the FMN preparationsused3 molesof NADPH for every mole of sulfide
1573
NADPH
--) FAD --) FMN MATERIALS
AND
--) heme + sulfite METHODS
Materials-Horse heart cytochrome c (type VI), FMN, GSH, p-CMPS,l and glucose oxidase (type V) were purchased from Sigma; benzyl viologen, DCIP, and FAD from Mann; NADPH, NADPf, NADH, and AcPyADP+ from P:L Biochemicals; methyl viologen from K and K Laboratories; menadione from Calbiochem; sodium arsenite from Mallinckrodt ; and Sephadex G-25 (coarse) from Pharamcia. NaHSOs, KNO*, NH%OH.HCl, and K,Fe(CN)s were Baker “Analyzed” reagents. CO, Hz, and Nz were purchased from Matheson; the latter two gases were freed of residual oxygen before use by passage through a column of hot copper. PAPS was prepared by the method of Kredich (11). E. coli sulfite reductase was purified by the procedure of Siegel et al. (6) ; all enzyme samples used in this study had a specific activity of at least 2.8 units 1 The abbreviations used are: p-CMPS, p-chloromercuriphenylsulfonate, monosodium salt; AcPyADP+, 3-acetylpyridine adenine dinucleotide phosphate; AcPyADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate; DCIP, 2,6-dichloroindophenol; MVH, reduced methyl viologen; PAPS, adenylyl sulfate3-phosphate.
per mg. Naz a5S03, specific activity 15 Ci per mole, was purchased from New England Nuclear. Enzyme Assays-NADPH-dependent reduction reactions were measured in l.O-ml reaction volumes containing 0.1 M potassium phosphate buffer (pH 7.7), 0.2 mM NADPH, acceptor. and an appropriate amount of enzyme. Acceptors were present at the following concentrations: sulfite, 0.5 mM; nitrite or hydroxylamine, 10 mM; oxygen, 0.25 mM; ferricyanide, menadione, FMN, FAD, DCIP, or cytochrome c, 0.1 mM; AcPyADP+, 0.2 mM. Rates were measured spectrophotometrically using a Cary model 14 spectrophotometer, with a control solution which for most reactions contained buffer in place of electron acceptor in the reference cuvette; for the NADPH-cytochrome c, DCIP, and AcPyADP+ reductase reactions, in which reduction of acceptor rather than oxidation of NADPH was measured, the control solution contained buffer in place of enzyme. NADPHferricyanide reductase and NADPH oxidase activities were also corrected for the nonenzymatic reaction. Absorbance changes were followed at 340 nm for all acceptors except the following: cytochrome c, (550 nm) ; DCIP, (600 nm); AcPyADP+, (363 nm). MVH-dependent reduction reactions were measured under anaerobic conditions in Thunberg cuvettes fitted with serum caps. Reaction mixtures contained, in 2.5 ml total volume, 0.1 1~ potassium phosphate buffer (pH 7.7), 0.1 mM MVH, acceptor (0.2 mM sulfite or NADP+), and an appropriate amount of enzyme. Buffer and acceptor, in a 2.3.ml volume, were added to the main compartment of the Thunberg cuvette, and 0.1 ml of enzyme was added to the side arm. The system was bubbled with On-free Nz for 15 min. The enzyme was then tipped in and 0.1 ml of MVH (reduced with H2/Pt asbestos) was added with a gas-tight Hamilton syringe to start the reaction. Control mixtures contained buffer in place of electron acceptor. Rates of MVH oxidation were measured spectrophotometrically at 604 nm using a Cary model 14 spectrophotometer. The following extinction coefficients were used: NADPH, 6.22 x lo3 M-’ cm-l (12) ; reduced minus oxidized cytochrome c, = 2.1 X lo4 M-I (13); DCIP, eeoo = 2.1 X lo4 M-I cm-l Ae55o (14); AcPyADPH minus NADPH, Aeac3 = 5.6 x 10s M-I cm-l (15); MVH, t604 = 1.14 X lo4 M-I cm-i (16). Unless otherwise indicated, rates were expressed as moles of NADPH (or 2 x moles of MVH) oxidized per mole of enzyme per min. Other Assays-Concentration of sulfite reductase was determined spectrophotometrically, using an extinction coefficient for the enzyme of 3.1 x lo5 M-I cm-r at 386 nm (6). Protein was measured by the Zamenhof (17) adaptation of the microbiuret method described previously (6). Sulfide and sulfite were measured by the methods of Siegel (18) and Grant (19), respectively; concentrations of standard solutions were determined by iodometric titration. FMN and FAD were measured fluorometrically by the procedure of Faeder and Siegel (20) ; concentrations of standard solutions were determined spectrophotometrically by means of their absorbances at 450 nm, utilizing reported extinction coefficients (1.22 X lo4 Me’ cm-l for FMN and 1.13 x lo4 Me1 cm-’ for FAD (21, 22)). Spectroscopic illeusurements-Absorption spectra were measured, tiersus appropriate solvent blanks, with a Cary model 14 spectrophotometer equipped with 0 to 0.1 A and 0 to 1.0 A slide wires. Fluorescence spectra were measured in a Turner model 210 spectrophotofluorometer, equipped with constant energy attachment. For determination of flavin concentrations, an excitation wavelength of 450 nm (band width 10 nm)
Downloaded from www.jbc.org by guest, on July 25, 2011
He also noted that an NADPH-hydroxylamine reductase activity copurified with the sulfite reductase. Lazzarini and Atkinson (4) further purified this enzyme as a NADPHnitrite reductase, and Kemp et al. (5) in Atkinson’s laboratory subsequently showed that the NADPH-sulfite, nitrite, and hydroxylamine reductions were catalyzed by a single enzyme. These workers showed that all three activities were inhibitable by arsenite and the mercurical p-chloromercuribenzoate as well as by cyanide; furthermore, the enzyme preparation contained a NADPH-cytochrome c reductase activity which copurified with the other activities cited, and which was repressed in cysteine-grown organisms. We have purified to homogeneity the NADPH-sulfite reductase of E. coli (6) and shown it to be a complex hemoflavoprotein of molecular weight 670,000. The enzyme contains, per mole, the following prosthetic groups: 4 FAD, 4 FMN, 20 to 21 atoms of iron, 14 to 15 labile sulfides, and 3 to 4 moles of a novel type heme. This heme has been identified (7) as an octacarboxylic iron-tetrahydroporphyrin of the isobacteriochlorin type (adjacent pyrrole rings reduced), and has now been observed to serve as prosthetic group of several sulfite reductases, both assimilatory and respiratory (8, 9). It has been termed “siroheme” (8). It is our object to describe the catalytic mechanism whereby a 6-electron reduction is accomplished by this hemoflavoprotein, one of the most complex arrays of electron-transport prosthetic groups yet observed in a single enzyme. To this end, we have investigated the interaction of sulfite reductase with a variety of electron donors, acceptors, and inhibitors, and have studied the effect of these agents both upon catalysis and upon optical properties of the enzyme. The results reported in this paper, some of which have been presented previously in preliminary form (lo), support the following conclusions: (a) The site of entry of pyridine nucleotide electrons is probably FAD. (5) The site of interaction of sulfite with enzyme appears to be the heme. (c) The FMN prosthetic group is required for electron transfer between the reduced FAD and the heme. These studies have not as yet assigned a specific role for the non-heme iron-labile sulfide groupings. The following minimum scheme is indicated :
1574
RESULTS
TABLE
-
Experi. merit
NADPH oxidized
Time
T
-
1
Sulfide produced
Sulfite reduced
NADPH/S’
N,$?8
--
Sulj2e Reduction Stoichiometry-As shown in Table I, E. coli sulfite reductase catalyzes the stoichiometric reduction of sulfite to sulfide at the expense of 3 NADPH, this stoichiometry being maintained For these measurements, throughout the course of the reaction. a reaction mixture containing NADPH, sulfite, and enzyme was incubated for varying periods during which t.he amount of NlZDPH oxidized was followed by the absorbance change at 340 nm. The reaction was stopped by addition of the colorforming reagents for determination of either sulfite or sulfide. Anaerobiosis was maintained to prevent oxygen-dependent consumption of NADPH (due to the NADPH oxidase activity of the enzyme, vide infra) and thereby avoid a spuriously high NADPH-sulfide stiochiometry. The results demonstrate that purified E. coli sulfite reductase can catalyze the complete 6electron reduction of sulfite to sulfide without the accumulation of significant quantities of sulfur-containing compounds of intermediate oxidation states. This behavior is in marked contrast to that reported for the dissimilatory sulfite reductases of Desulfovibrio (24, 25) and Desuljotomaculum (26), which appear to catalyze an incomplete reduction of sulfite to sulfide, with an observed stoichiometry of 10 to 12 electrons consumed per sulfide produced. With the Desulfovibrio enzyme, sulfurcontaining intermediates such as trithionate and thiosulfate have been reported to accumulate in the reaction mixture during the course of sulfite reduction (24-30). Kinetic Parameters-A series of Lineweaver-Burk plots of the initial velocities of NADPH oxidation at varying sulfite and NADPH concentrations is shown in Fig. 1. From these data, of sulfite reduction, at “infinite” concentrations of the V,,, both reactants, is 1850 NADPH per enzyme per min in 0.1 M potassium phosphate buffer, pH 7.7, at 23”. The K, for sulfite, at infinite concentration of NADPH, is 4.3 PM. The K, for NADPH, at infinite concentration of sulfite, is 4.5 PM (Table 11). These values are somewhat lower than those reported previously (sulfite K, = 7 to 9 /JM, NADPH K, = 18 to 60 ).kM (5, 31)), but are considered more reliable, since the present
I
Stoichiometry of NADPH-dependent sul$te reduction Reaction mixtures contained in a 3.0-ml total volume: 0.1 M potassium phosphate, pH 7.7; 200 GM NADPH; 80 PM NaHS03; 25 nM sulfite reductase; 10 units per ml of glucose oxidase; and 10 mM glucose. The mixtures were present in anaerobic cuvettes (stoppered with tight-fitting serum caps) of l-cm light path and reactions were initiated by injecting Nz-bubbled solutions containing all substrates with 5 ~1 each of glucose oxidase and sulfite reductase, in succession, with a period of 60 s between injections. NADPH oxidation was followed by the decrease in absorbance at 340 nm with a Cary model 14 spectrophotometer. Absorbance readings were initiated approximately 10 s after injection of sulfite reductase, and the AA340 extrapolated back to time of injection. At each of the indicated times, the reaction was stopped by addition of the color-forming reagents used in determination of sulfide (Experiment 1) or sulfite (Experiment 2). The amount of sulfite in each reaction mixture was subtracted from the amount present in a control reaction mixture from which sulfite reductase was omitted. The amount of sulfide in each reaction mixture was determined with reference to a control in which sulfite reductase was omitted. There was negligible nonenzymatic disappearance of sulfite or production of sulfide during the time period of the assay. NADPH oxidation was also negligible in a control sample from which sulfite had been omitted.
min
nmoles
2 4 6 8 12 16
134 245 336 400 493
20
550
8
-
-
2.91 3.02 2.97 2.90 3.06 3.02 3.02
81 113 138 161 179 182
540
2 4 6 12 16 20
46
143 242 325 400 488 531 539
48 82 106 140
-
-
2.98 2.95 3.07 2.86 3.05 2.97
169 179 180
2.99
-
measurements were obtained with 5- and IO-cm light paths and a spectrophotometer with a 0 to 0.1 A slide wire, where necessary, to facilitate measurement with substrate concentrations in the 1 to 10 PM range. All previous data were obtained using l-cm light paths. The fact that the reciprocal plots yield parallel lines is compatible with (but does not require) a catalytic mechanism in which the first reactant, presumably NADPH, converts enzyme to a reduced form which subsequently interacts with an oxidizing substrate to yield original enzyme and final product (32). This is compatible with the previously noted (6, 10) reduction of enzyme by NADPH in the absence of acceptor, as deduced from optical and EPR spectroscopy. pH Optimum-When the velocity of NADPH oxidation was studied as a function of pH, using the standard assay concentrations of NADPH and sulfite, the optimum pH was 7.9 (Fig. 2). Activities were identical in 0.1 M potassium phosphate and Tris-HCl buffers. Since the pK, for HS03 is 7.2 (33), the predominant sulfite species in solution at the optimal pH is soag-. Electron Donors-In addition to NADPH, sulfite reductase
Downloaded from www.jbc.org by guest, on July 25, 2011
and an emission wavelength of 535 nm (band width 25 nm) were utilized. Fluorescence polarization measurements were made with a Farrand Mark II spectrophotofluorometer. All spectroscopic measurements were performed at 23-25”, utilizing l-cm light paths unless otherwise indicated. Radioactivity Measurements-Radioactivity of a5S-containing solutions was determined on appropriately diluted aliquots (4 ml of aqueous sample plus 16 ml of the xylene-Triton X-114 mixture of Greene (23), with the naphthalene omitted) with a Packard model 3375 Tri-Carb liquid scintillation spectrometer. For all samples of standards and unknowns, measurement of radioactivity was continued until the statistical counting error was less than 1 To. Concentration and Gel FiZtration-Ultrafiltration of enzyme solutions was performed with an Amicon concentrator equipped with a Diaflo PM-30 membrane. For removal of low molecular weight solutes from enzyme in ligand-binding experiments, l.Oml samples were applied to a column (1.5 X 15 cm) of Sephadex G-25 (coarse) and 1.2.ml fractions were collected. Following either concentration or gel filtration, enzyme content was determined in appropriate fractions by measurement of protein concentrat.ion. In all such experiments, recovery of enzyme protein was at least 85%.
1575
2.5
PH
2. pH optima of the NADPH-sulfite, -nitrite, and -hydroxylamine reductase reactions. Reaction rates were measuredas indicated under “Materials and Methods,” with either potassium phosphate (circles), Tris-HCl (triangles), or glycine-KOH (squares),all at 0.1 M and at the pH valuesindicated, substituted for the standard assay buffer. FIG.
0
I
I 1
I 2
3
4
‘k,LFITEI
x lo5
5
6
II
TABLE
Kinetic
parameters fofop some NADPH-dependent catalyzed by Escherichia coli sul$te
reactions
reductase
Kinetic parameters were determined from Lineweaver-Burk plots in reaction mixtures containing 0.1 M potassium phosphate (pH 7.7) as buffer and 1 m FMN (34). K,,, values were obtained by extrapolation of the plots to infinite concentration of the second substrate. Vmax values represent rates at “infinite” concentration of both substrates. -
-
Acceptor
Form of plot
V max
I
Sulfite.. . Nitrite. . Hydroxylamine. Cytochrome c. . Ferricyanide. .. DCIP. . Menadione .
.-
NADPH/ min/e?my?nc
Parallel lines Parallel lines Convergent lines Parallel lines Parallel lines Parallel lines Parallel lines
1,850 3,100 13,700
4.3 4.5 800.0 26.0 1’0,300.o 53.0 36,000 7.5 , 35.0 37,000 11.0 38.0 40,000 26.0 67.0 39,000 81.0 80.0
can utilize reduced methyl and benzyl viologens as electron donors
for sulfite
reduction.
The
product
in both
cases is
sulfide. As shown in Table III, the velocity with MVH (0.1 mM) is about twice that observedwith NADPH (0.2 mM). No variation of the rate of MVH oxidation was observedwhen the concentration of MVH was varied in the range 20 to 200 PM. Similarly, the reaction velocity with MVH as electron donor was independent PM.
of sulfite concentration
in the range 20 to 500
Reducedbenzyl viologen (0.1 mM) is a lesseffective donor,
yielding
a reaction
velocity
13ye of that observed with methyl
viologen at the same concentration of substrates. NADH, FMNH2, GSH, and reduced cytochrome c, all at 0.2 mM concentration, did not promote conversionof sulfite to sulfidewith enzyme sufficient to allow detection of a reduction rate 1% of that found with NADPH aselectron donor. Othm ReactionsCatalyzed
In addition to sulfite reduction, E. coli sulfite reductaseis capable of catalyzing a number of other pyridine nucleotidedependentreduction reactions. NADPH-Nitrite and Hydroxylamine Redmtase-As reported previously (4), E. coli sulfite reductasecatalyzes NADPH-dependent reduction of hydroxylamine and nitrite to ammonia. MVH can also serve as electron donor for reduction of these substrates,but we have not studied this reaction quantitatively. Lineweaver-Burk plots of the initial velocities of NADPH oxidation at varying nitrite and NADPH concentrationsyield a seriesof parallel lines,as wasobservedwith sulfite as acceptor. With hydroxylamine as electron acceptor, on the other hand, a seriesof converginglinesis obtained; we have no ready explanation for this observation. Kinetic parametersfor the NADPH-nitrite and -hydroxylaminereduction reactionsare presentedin Table II. The Ir,,, valuesfor both nitrite (3100NADPH per enzyme per min) and hydroxylamine (13,700NADPH per enzymeper min) reduction are greater than that observedwith sulfite, but the K, values for thesesubstrates(0.8 mMfor nitrite, and 10mM for hydroxylamine) are much higher than for sulfite (4.5 PM). The pH optima for nitrite and hydroxylamine reduction, 8.6 and 9.5, respectively, are more alkaline than that for sulfite reduction (7, 9) (Fig. 2). As shownby Kemp et al. (5), it is unlikely that NADPH-sulfite reductasefunctions physiologically as a nitrite or hydroxylamine reductase. NADPH-Diaphmase and MVH-NADP+ Reductase ActititiesSulfite reductasecatalyzesthe transfer of electronsfrom NADPH to a wide variety of acceptors,including cytochrome c, ferricyanide, DCIP, menadione,and FMN. As shownin Table III, the rates of these diaphorase-type reactions, under standard assayconditions(0.2 mMNADPH and 0.1 mMacceptor), varied from 10,000 to 28,000 NADPH per enzyme per min. FAD
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1. Lineweaver-Burk plot of NADPH-sulfite reductase activity as a function of sulfite concentration. Reaction mixtures contained 0.1 M potassium phosphate (pH 7.7), 0.3 to 1.2 nM enzyme, and the indicated concentrations of NADPH and sulfite. Absorbance change was followed at 340 nm in a Cary model 14 spectrophotometer equipped with 0 to 0.1 A and 0 to 1.0 A slide wires, at 23” in cells of either 5- or lo-cm path length. The reference cuvette contained buffer in place of sulfite. Initial velocities (v) are expressed as moles of NADPH oxidized per mole of enzyme per min. The points at infinite concentration of NADPH were obtained from the intercepts of a l/v sers’susl/(NADPH) plot, at several sulfite concentrations, of the same data plotted in the figure. Such plots also yielded a series of parallel lines. FIG.
1576 III
TA~LIC
catalyzed by Escherichia coli Reactions were measured as described under “Materials and Methods.” Rates are expressed as a-electron equivalents transferred per enzyme per min. With NADP’, p-CMPS, and fluoride &s inhibitors, enzyme was incubated in 0.1 M potassium phosphate buffer (pH 7.7) containing the inhibitor for the period of time indicated below, and the reaction initiated by addition of electron acceptor and NADPH. With cyanide, CO, and arsenite, an anReactions
-
rurnover
Reaction
number
I
sul$te
reductase:
e$ect
of inhibitors
aerobic solution of enzyme plus NADPH was incubated with the inhibitor and the reaction initiated by addition of electron acceptor. Activities are expressed relative to a control treated in parallel in which buffer replaced the inhibitor solutions. Incubation times: NADP+, cyanide, arsenite, fluoride, and 0.2 rnM p-CMPS, 5 min; 1 pM p-CMPS, 60 min; CO, 30 min. Per
cent of control activity in presenceof
NADP+ (10 nIY)
--
CN(1 mu)
p-CMPS (0.2 rind)
(OEM,
AS025 (10 mar)
(0 f-M)
Ze-/eneyme/min
+ --f ---) --* --f -+ -+ -+ + -+ -+ -+
AcPyNADP+. . FMN DCIP.. Fe(CN)sa-. Menadione. . Cytochrome c. . 02. . NADP+. Sulfite. . . Nitrite.. Hydroxylamine Sulfite. .
9,500
100
12
10,300
85 17 14
10 15 11 13 0
27,700 28,100
19,100
-
27,500 75 36,300 1,800 2,600 5,900 3,900
10 1 4 5 5 2
4 5 5 -b
-
-
1 105
5 0 5 0 0 103
-
102 93 99 102
104 99 109 107
91
100 104
97 96 95 4 3 0 0
94 -a 8
10 12 10
102 96 98 97 92 98
104
91
100 114 95 110 102 108
87 21 15 9 18
98 28 68 51 27
-
a Incubation of enzyme with MVH for 30 min causes 90% inhibition of MVH-NADPH+ reductase activity (but not MVH-sulfite reductase activity). Therefore, inhibition of this activity by CO, which requires prolonged incubation with CO in the presence of reductant, was not examined. b Not examined because of high rate of MVH-NADP+ reductase activity catalyzed by sulfite reductase. (0.1 mM) also served as an acceptor for the electrons of NADPH, with a velocity of 5,600 NADPH per min per enzyme. Kinetic studies of four of these diaphorase-type reactions are summarized in Table II. Each of the reactions studied, i.e. t,he NADPHdependent reductions of cytochrome c, ferricyanide, DCIP, and menadione, yielded a series of parallel lines in Lineweaver-Burk p1ot.s. Although K, values for NADPH and acceptor varied with the reaction studied, the V,,, values, at infinite concentrations of both NADPH and acceptor, were identical within experimental error for each of the four reactions (38,000 f These turnover numbers 2,000 NADPH per enzyme per min). represent the highest observed for any of the reactions catalyzed by sulfite reductase, and are over 20 times as great as the V,,, for sulfite reduction with NADPH as electron donor. The results suggest the presence of a common rate-limiting step in the reductions of cytochrome c, ferricyanide, DCIP, and menadione. An additional rate-limiting step, considerably slower than that for reduction of diaphorase-type acceptors, must become operative in the reduct)ion of sulfite. The enzyme also catalyzes another diaphorase-type reaction, the reduction of methyl viologen by NADPH. Since the potential of MVH is considerably more negative than that of NADPH, we have followed the reverse reaction, i.e. the reduction of NADP+ by MVK. The observed velocity of the latter reaction, 36,000 NADPH per enzyme per min (Table III), is comparable to that of the other diaphorase activities of sulfite reductase. NADPH Oxidase-Sulfite reductase can catalyze the oxygendependent oxidation of NADPH (Table III). At 0.2 mM NADPH and 0.25 mM 02, this reaction proceeds with a velocity of 75 NADPH per min per enzyme, i.e. 4% of the rate of the NADPH-sulfite reductase activity in the standard assay. No
detailed studies of the NADPH oxidase activity have been performed. Transhydrogenase-Sulfite reductase NADPH-AcPyADP+ catalyzes t.he transfer of hydrogen from NADPH to AcPyADP+ (Table III). At 0.2 mM concentration of each nucleotide, the reaction velocity is 9500 NADPH per min per enzyme. When velocity is plotted versus concent.ration of either substrate, at a fixed concentration of the other, the curve exhibits a maximum, indicating inhibition by excess substrate. Detailed kinetic analyses of the transhydrogenase reaction (as well as competitive inhibition by NADP+ (vi& infra)) are compatible with a common binding site for both oxidized and reduced pyridine nucleotides. Other Electron Acceptors Tested-None of the following compounds stimulated the anaerobic oxidation of NADPH, under conditions in which an activity 1 y0 of that observed with sulfite as acceptor could have been easily measured: Na2S04 (10 nM), Na&&03 (1 mM), Na&06 (1 mM), PAPS (0.1 mM), NanSeOn (1 mM), NaAsOz (10 mM), NaN03 (10 mM), hydrazine hydrochloride (10 mM), NaNa (10 mM). Inhibitors By studying the effect of inhibitors on the various reactions catalyzed by E. co& sulfit,e reductase, we hoped to define more clearly those segments of the enzyme molecule with which different electron donors and acceptors can interact, and thereby tentatively deduce a sequence of electron flow within the sulfite reductase hemoflavoprotein molecule. CO and Cyanide-We first examined the catalytic effects of inhibitors which can be expected to react with the heme moiety, These compounds have been demonstrated i.e. CO and KCN. (6) to react with both free and enzyme-bound sulfide reductase
Downloaded from www.jbc.org by guest, on July 25, 2011
NADPH NADPH NADPH NADPH NADPH NADPH NADPH MVH NADPH NADPH NADPH MVH
1577 heme to form spectrally distinct complexes. As reported (6), CO can complex only to reduced heme, while cyanide can be a ligand to either reduced or oxidized heme. However, since only the reduced enzyme is “accessible” to cyanide, the oxidized enzyme-cyanide complex can only be observed by first preparing the reduced enzyme-cyanide complex, and then permitting it to oxidize. The enzyme-cyanide complex forms rapidly but apparently irreversibly (6) ; the enzyme-CO complex forms reversibly, but its rate of formation and dissociation is slow. The subsequent section will describe the correlation between the spectrophotometrically observed processes of enzyme hemeligand complex formation, and the catalytic events which are presumed to involve the heme. When sulfite reductase was incubated with CO or cyanide in the presence of reductant, as described in Table III, the NADPHsulfite, nitrite, and hydroxylamine reductase activities were substantially inhibited, as was the reduction of sulfite by MVH. In contrast, none of the NADPH-diaphorase, oxidase, or trans-
indicated,
in a total
volume
At the times
of 0.9 ml.
indicated,
the
NADPHsulfite reductase reaction was initiated by addition of 0.1 ml of 5 mM NaHSOa (when NADPH was present in the preincubation mixture) or 0.1 ml of a solution containing 5 mM NaHSOa plus 2 mM NADPH (when NADPH was not nresene in the nreincubation mixture). All‘solutions were in 0.1 i potassium phosphate (pH 7.7). Absorbance changes were followed at 340 nm with a Cary model
14 spectrophotometer
at 23”.
FIG. 4 (right). Kinetics
of enzyme-CO
development of inhibition of NADPH-sulfite CO. Formation of enzyme-CO complex,
taining
5.36 pM enzyme, 0.5 mM NADPH,
complex formation
and
reductase activity C---C : a solution
by con-
0.5 mM CO, and 0.1 M
potassium phosphate (pH 7.7), in a total volume of 1.0 ml, was incubated under anaerobic conditions at 23” and successive absorption spectra recorded (versus a blank containing buffer alone).
The amount of enzyme-CO complex present at any time (i) is proportional to the difference in absorbance of the enzyme solution at the wavelengths 609 nm and 560 nm (6). The reaction was followed for 90 min, and the tis~-560 value at t = m represents the value at that time. Development of CO-inhibition of NADPH-
sulfite reductase activity, O---O : a solution containing 20 nm enzyme, 0.2 mM NADPH, 0.5 mM CO, and 0.1 M potassium phosphate (pH 7.7), in a total volume of 0.9 ml, was incubated, in a cuvette of l-cm path length, under anaerobic conditions at 23’for the time
indicated.
The
NADPH-sulfite
reductase
reaction
was then
initiated by addition of 0.1 ml of 5 mM NaHS03 to the cuvette containing the enzyme-NADPH-CO solution. Absorbance changes were followed at 340 nm with a Cary model 14 spectrophotometer at 23”. Inset, dependence of pseudo-first order rate constant for inhibition tration.
of NADPH-sulfite reductase The kinetics of develonment
activity upon CO concenof inhibition of sulfite re-
ductase activity was measured as described above with each of the CO concentrations
indicated.
* A different type of experiment, previously reported (lo), had also indicated a close relationship between spectrophotometric and catalytic data. In that experiment, CO was added to an NADPHcontaining enzyme solution under conditions of incomplete anaerobiosis, so that two successive treatments with reductant were
required
before
complex
formation
was complete.
The
time
course of appearance of spectral changes was therefore irregular. The irregularities in development of spectral changes were parallelled by corresponding irregularities in development of inhibition of sulfite reductase activity.
Downloaded from www.jbc.org by guest, on July 25, 2011
TIMEOF INCUIATIONw,m co (no”,
PRElNCUBATlON TlME (mm)
FIG. 3 (left). Inhibition of NADPH-sulfite reductase activity anaerobically by CO and cyanide. Enzyme, 20 nM, was incubated at 23” with 0.5 mM CO, 50 PM KCN, and/or 0.2 mM NADPH, as
hydrogenase reactions was significantly inhibited by either CO or cyanide. The relationship between spectrophotometrically observable CO and cyanide binding to the heme prosthetic group and the inhibition of sulfite reductase activity was examined in detail as described below. The following correlations were obtained. 1. CO and cyanide bind only to reduced heme, and inhibition of activity by these agents occurs only if enzyme is reduced prior to addition of sulfite. As shown in Fig. 3, when CO or cyanide was incubated with enzyme (either with or without sulfite) in the absence of a reducing agent. and the remaining reactant(s) were added to start the sulfite reductase reaction, no inhibition of sulfite reductase activity, as compared to controls, was detected. However, when enzyme was incubated with NADPH plus either CO or cyanide, a progressive inhibition of sulfite reductase activity was observed. 2. The rate of CO and cyanide binding to the heme equals the rate of development of inhibition of sulfite reductase activity. The rate of CO binding to reduced enzyme was followed by recording at successive time intervals the absorption spectra of the enzyme plus NADPH plus CO solution. As described previously (6), the AAs,,o-aso of the enzyme solution is a good measure of the amount of enzyme-CO complex formed, since the AA between these two wavelengths is negligible in both oxidized and reduced enzyme, while the formation of the CO complex is accompanied by greatly increased absorbance at 600 nm with little change at 560 nm. Aliquots of the reaction mixture were measured periodically for NADPH-sulfite reductase activity, while AA600-560 was monitored. As shown in Fig. 4, the rates of formation of enzyme-CO complex, determined spectrophotometrically, and of loss of sulfite reductase activity, exhibited identical pseudo-first order kinetic patterns, with identical rate constants of 1.56 X 1O-3 s-l at 0.5 mM CO. This corresponds to a second-order rate constant of 3.1 M-I s-l for the reaction E + CO -+ E-CO, in agreement with that reported previously (6) for the formation of the E-CO complex. The rate of inhibition of sulfite reductase activity by CO was followed as a function of CO concentration. As shown in the inset to Fig. 4, the pseudo-first order rate constants for this process were proportional to CO concentration, and a second order rate constant of 3.2 M-l s? could be obtained from the This value is in excellent agreement with that slope of this line. measured for formation of the enzyme-CO complex.* The rate of cyanide binding to reduced enzyme has not been studied previously. A difference spectrum between reduced enzyme plus cyanide and reduced enzyme is shown in Fig. 5. A prominent maximum is observed at 411 nm. The timedependence for the development of this absorbance change was compared to that for development of cyanide inhibition. These data are shown in Fig. 6. The AAdu between the two solutions increased according to pseudo-first order kinetics. If one assumes that the rate of the reactions E + cyanide -+ E-cyanide, determined spectrophotometrically at the cyanide concentration indicated in Fig. 6, is in fact proportional to the product
1578 (Enz*m*
l
mi”“‘
(Enty..
l
KCNI
NADPH)
l
I
I
500
400
NADPH
I
600
WAVELENGTH
.
700
(inn)
FIG. 5. Difference spectrum between reduced enzyme-cyanide complex and reduced enzyme. The sample cuvette contained 3.5 PM sulfite reductase, 0.2 mM NADPH, and 0.1 mM KCN, in a total volume of 1.0 ml, while the reference cuvette contained 3.5 PM enzyme and 0.2 mM NADPH, but no KCN. All solutions were anaerobic and in 0.1 M potassium phosphate (pH 7.7). The difference spectrum was recorded, approximately 5 min after addition of KCN to the reduced enzyme in the sample cuvette, with a Cary model
14 spectrophotometer
at 23” in cells of l-cm
path
5 TIME
FIG.
6. Kinetics
OF
INCUBATION
WITH
KCN
of enzyme-cyanide
(min)
complex
formation
and de-
velopment of inhibition of NADPH-sulfite reductase activity by cyanide. Formation of enzyme-cyanide complex, 0-C: two cuvettes (fitted with rubber serum stoppers) were prepared, each of which contained an anaerobic solution of 3.5~~ sulfite reductase and 0.2 mM NADPH
in a total
volume
of 1.0 ml.
To the reference
0, 4’ Ihourr)
TIME
FIG.
7. Dissociation
of NADPH-sulfite
of enzyme-CO
reductase
activity.
complex and reappearance Enzyme-CO
complex
was
cuvette, 5 ~1 of buffer were added. To the sample cuvette, 5 ~1 of 20 my KCN were added, and the change in absorbance at 411 nm between the two cuvettes recorded with a Cary model 14 spectrophotometer. All solutions were in 0.1 M potassium phosphate (pH
formed by incubating 15 FM sulfite reductase anaerobically with 0.2 rnM NADPH and 0.5 mM CO at 23” for 1 hour. The l-ml solution was then passed through a column of Sephadex G-25 as described under “Materials and Methods,” and the resulting en-
7.7),
zyme, 5.2 PM by protein determination, was incubated at 4’. At the times indicated, absorption spectra of the solution were re-
in cells of l-cm
path
length,
at 23”.
The
amount
of enzyme-
cyanide complex present at any time (t) is proportional to the AAdrr between the two cuvettes at that time. The reaction was followed for 10 min, and the AA,rr value at t = m represents the value at that time. Development of cyanide inhibition of
corded (at 23”) and aliquots assayed for NADPH-sulfite reductase activity. A control sample of enzyme was treat,ed with 0.2 mM NADPH anaerobically, passed through the Sephadex G-25 column,
NADPH-sulfite
and
reductase
activity,
O---O
: a solution
containing
20 nm enzyme, 0.2 mM NADPH, 0.1 mM KCN, and 0.1 M potassium phosphate (pH 7.7), in a total volume of 0.9 ml, was incubated at 23” for the time indicated in a l-cm path length cell. The NADPH-sulfite reductase reaction was then initiated by addition
incubated
at 4” in
parallel
with
the
enzyme-CO
complex.
NADPH-KCN solution. Absorbance changes were followed at 340 nm with a Cary model 14 spectrophotometer at 23’. Inset, dependence of pseudo-first order rate constant for inhibition of NADPH-sulfite reductase activity upon KCN concentration.
Aliquots of the latter solution were assayed for sulfite reductase activity each time the enzyme-CO solution was so assayed. The activity of the control enzyme solution decayed by only 10% during the entire period of incubation at 4”. Fraction of enzyme-CO complex remaining, determined as AAW-XQ at time 1/AAm-56o at the beginning of the incubation at 4” (to), C-0. NADPHsulfite reductase activity of enzyme-CO solution (expressed as per cent of control solution, corrected to equivalent protein concentrations), O-O. Inset, kinetics of dissociation of enzyme-CO
The
complex
of 0.1 ml of 5 mM NaHS03
kinetics
of development
to the cuvette
of inhibition
containing
of sulfite
the enzyme-
reductase
ac-
tivity was measured as described above with each of the KCN concentrations indicated.
(0-C)
and reappearance
of NADPH-sulfite
reductase
activity (O-O). The activity after 105 hours of incubation 4” (7Oyc of control enzyme) was taken as the t = 00 value.
at
Downloaded from www.jbc.org by guest, on July 25, 2011
O.OllI
length.
(E) . (cyanide), then a second-order rate constant of 210 .M+ s-l can be calculated. The rate of development of inhibition of sulfite reductase activity by cyanide in the presence of NADPH was then compared to the rate of formation of the E-cyanide complex. At each of the cyanide concentrations tested, the loss in activity followed pseudo-first order kinetics (Fig. 6). As shown in the inset to Fig. 6, the pseudo-first order rate constants for cyanide inhibition of sulfite reductase activity were proportional to cyanide concentration, yielding a value for the second order rate constant for the cyanide inhibition of sulfite reductase activity of 201 M-r 0, in good agreement with the value obtained from spectrophotometric measurements for formation of the E-cyanide complex. 3. The rate of dissociation of the enzyme-CO complex equals the rate of reappearance of sulfite reductase activity. A solution of enzyme-CO complex was prepared as described in Fig. 7 and maintained at 4”. Aliquots were examined at intervals over a 5-day period for content of enzyme-CO complex (AAc~~-w,) and for sulfite reductase activity. The results are shown in Fig. 7. As CO dissociated from the complex to yield free, oxidized enzyme (6), sulfite reductase activity reappeared. Both reactions followed first-order kinetics with the same rate constant: 1.2 x
1579 (NADPH). When the reduced complex was mixed with air and allowed to reoxidize, the spectrum returned to that of native, oxidized enzyme. Similarly, if the reduced enzyme-arsenite complex was passed through a column of Sephadex G-25 (aerobically) to remove NADP(H) and excess arsenite, the recovered enzyme was spectrally indistinguishable from free oxidized enzyme. This result, together with data on inhibition of enzyme activity to be presented below, suggests that arsenite can form a stable complex only with reduced enzyme; the complex dissociates rapidly when the components required for its formation are removed. With the concentrations of arsenite tested (1 and 10 mM), complex formation with NADPH-reduced enzyme, as measured spectrophotometrically, was complete within 10 s, the minimum time required to initiate measurement in the Cary model 14 spectrophotometer. In the experiments to be described, preincubations of enzyme with arsenite or other components, or both, were routinely conducted at 23” for 1 min, but identical results were obtained when preincubations with arsenite were as short as 10 s or as long as 20 min. When sulfite was added to preformed reduced enzyme-arsenite complex (enzyme preincubated with arsenite plus NADPH), the initial rate of sulfite reduction was strongly inhibited (Fig. 9, Curve A). However, the reaction rate progressively increased until a steady state constant rate of about
I
[AsOil
80
300
400
500 WAVELENGTH
600 (nm)
7100 TIME
AFTER
ADDITION
8 (left). Spectra of sulfite reductase in presence of arsenite. The following additions were made to an anaerobic solution of 2.7 I.~Menzyme in 0.1 M potassium phosphate buffer (pH 7.7), and absorption spectra were recorded as soon as possible after addition of all components with a Cary model 14 spectrophotometer at 23” in cells of l-cm path length: A, no addition; B, 10 mM NaAsO, (superimposable upon A), p; C, 0.5 mM NADPH, ..*a ; D, 10 mM NaAsOz plus 0.5 rnM NADPH, -----. FIG. 9 (center). Effect of order of addition of components upon arsenite inhibition of NADPH-sulfite reductase activity. Reaction mixtures contained, in 1.0 ml total volume, 8 nM enzyme, 0.2 mM NADPH, 0.5 mM NaHS03, and 5 mnn NaAsOz where indicated. The indicated components were preincubated for 1 min in a volume of 0.9 ml. The final component(s) was then added in a volume of 0.1 ml and the absorbance change at 340 nm followed in a Cary model 14 spectrophotometer with respect to a reference solution which contained all components except sulfite. All solutions were in 0.1 M potassium phosphate (pH 7.7), cells were 1 cm in path length, and all operations were performed at 23”. Each curve represents the average of three independent measurements. A, FIG.
OF
LAST
COMPONENT
Irec)
preincubation: enzyme plus arsenite plus NADPH; final component: sulfite. B, preincubation: enzyme plus amenite; final component: sulfite plus NADPH. C, preincubation: enzyme plus arsenite plus sulfite; final component: NADPH. D, preincubation: enzyme plus sulfite plus NADPH; final component: arsenite. E, preincubation: sulfite plus NADPH plus arsenite; final component: enzyme. Controls, same as A through E, except arsenite omitted. There was no significant difference in the control curves for A through E. FIG. 10 (right). Inhibition of NADPH-sulfite reductase activity by arsenite. Reaction mixtures contained 0.1 M potassium phosphate (pH 7.7), 9 nM enzyme, 0.2 mM NADPH, and the indicated concentrations of NaAsOz and sulfite. Reactions were initiated by the addition of sulfite as the final component. Absorbance change was followed at 340 nm in a Cary model 14 spectrophotometer at 23” in cells of l-cm path length. Rates (v) were determined from the linear portion of the progress curve, following a short, (
