ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

170,

514-528

(197%

The Properties of Sulfite Oxidation in Perfused Rat Liver; Interaction of Sulfite Oxidase with the Mitochondrial Respiratory Chain’ NOZOMU Johnson

Research

Foundation, University

OSHIN02

AND

BRI’M’ON

CHANCE3

Department of Biophysics and Physical Biochemistry, of Pennsylvania, Philadelphia, Pennsylvania 19174 Received

March

School

of Medicine,

14, 1975

Sulfate oxidation in the isolated mitochondrial fraction of rat liver proceeds exclusively through the respiratory chain in a sequence of electron flow from sulfite oxidase to cytochrome c and then to cytochrome oxidase. Thus, the SO,*-/0 ratio observed is almost unity and the ATPIO ratio with sulfite is half that obtained with succinate as the respiratory substrate. Direct reduction of molecular oxygen to H,O, by sulfite oxidase occurs only when the respiratory chain is inhibited by cyanide. These two reactions differ with respect to the effects of the O2 concentration and the sulfite concentration on the activity and on the S0,2-/0 ratio. In the perfused liver, infusion of sulfite causes increased uptake of O,, with concomitant reduction of the mitochondrial components, such as pyridine nucleotides, flavoproteins, cytochrome c and cytochrome oxidase. The SO,‘-/0 ratio observed is 1.6-1.4 and no increase in H,O, production is detected during sulfite oxidation, indicating that sulfite oxidase is located between the outer and inner membranes of mitochondrion and in contact with cytochrome c, thus providing reducing equivalents to the respiratory chain. In the presence of excess cyanide, 0, consumption increases two-fold during sulfite oxidation but the rate of sulfite oxidation does not change significantly. Under these conditions the S0,2-/0 ratio is 0.8-0.6, indicating direct reduction of 0, to Hz02 by sulfite oxidase in the perfused liver. The production of H,Oz is accompanied by a remarkable oxidation of pyridine nucleotides which is attributed to the stimulation of the glutathione-peroxidase reaction. The maximal rate of sulfite oxidation is more than 1.2 pmol/min/g wet wt in the control liver, and decreases to 0.6 pmol/min/g wet wt in the liver of the tungsten-pretreated rat which possesses 23% of the sulfite oxidase activity. As the rate of sulfite oxidation reaches its maximum, progressive inhibition of some dehydrogenases appears to occur in the perfused liver.

outer and inner membranes of mitochondrion (3-5). Sulfite is a specific reductant for this enzyme (6-8). The oxidation of sulfite by purified sulfite oxidase proceeds at almost the same rate with cytochrome c or molecular oxygen as electron acceptor (6, 7). The reaction with cytochrome c (Eq. [l]) when coupled to the mitochondrial respiratory chain as in Eq. [21 may reduce molecular oxygen to water, whereas the product of oxygen reduction by sulfite oxidase (Eq. [31) is H,Oz (6, 7).

Sulfite oxidase is one of the metallohemoproteins having molybdenum and protoheme as prosthetic groups (1, 2). This oxidase can be extracted from intact mitochondria by hypotonic treatment or by detergent treatment, and it has been suggested, by analogy to adenylate kinase, that sulfite oxidase is located between the 1 This study was supported by US research grants USPHS-HL-15061, AA-00292 and GM-12202. 2 Present address: Research and Development Department, Nihon Schering K. K., Higashi-yodogawa, Osaka, Japan. 3 To whom correspondence should be addressed.

SO,*514

Copvright A11 &hts

0 1975 by Academic of reproduction

in any

Press, Inc. form

reserved.

+ H,O

+ 2 cytochrome c3+ + Sod2 + 2 H+ + 2 cytochrome

czc

[ll

SULFITE 2 cytochrome

OXIDATION

c*+ + i/z O2 -+ 2 cytochrome

SOzz-

+ Hz0

+ 02 + SO,*-

IN

c3+ + H,O

[2l

+ H,O,

131

The direct reduction of O2 by sulfite oxidase is prevented completely in the presence of ferric cytochrome c (7). In intact mitochondria, therefore, sulfite oxidation seems to take place through the interaction of sulfite oxidase with the respiratory chain, producing one ATP per sulfite oxidized (5, 9). It has been established that the oxidation of sulfite to sulfate is a physiological reaction in the terminal process of biological degradation of sulfur-amino acids (10, 11). Inhaled SOZ and injected bisulfite are mostly excreted as sulfate in the urine (12, 13). Evidence indicating the involvement of sulfite oxidase in this process has been provided by the observation that a complete deficiency of sulfite oxidase in a child was accompanied by a lack of sulfate excretion in the urine (14, 15). Furthermore, pretreatment of rats with tungsten under molybdenum-deficient conditions results in decreases in the content of sulfite oxidase and xanthine oxidase, which is accompanied by an increased susceptibility to bisulfite toxicity (16). The radical chain reaction of sulfite auto-oxidation (17-22) has been employed in various experimental systems as an indicator of 02- formation: SO,*-

+ Oz-

+ 3 H+ 4 HSO,

+ 2 OH

141

SOS*-

+ OH

+ 2 H+ + HSO,

+ Hz0

151

HSO, HS03

+ 02 + SO, + O,-

+ H+

+ OH + SO, + H,O 2 HSO,

SO, + H,O

+ SO, + SO,*-+ SO,*-

+ 2 H+

I61 171

+ 2 H+

161 [Ql

According to Yang (201, 02- and HS03 can initiate the chain propagation reactions of Eqs. 14-61, and sulfite oxidation is maintained through these reactions with the concomitant formation of sulfate in Eq. 17-91. The chain length of the reaction approaches 33,000 per 02- in the xanthinexanthine oxidase system (21) and 330 per 0~ in the isolated chloroplast fraction under illumination (25). In rat liver in situ,

PERFUSED

515

LIVER

the rate of Hz02 production is on the order of 0.4 pmol/min/g wet wt of liver (26). A portion of the H202 detected may arise from the formation of 02- with subsequent enzymic dismutation into HzOz. The inhibitory action of sulfite on various enzymes is noted; bisulfite is a potent inhibitor of malate dehydrogenase cKi = 5 PM) (27) and sulfite interacts with various flavoprotein oxidases such as glycolate oxidase, causing a bleaching of flavoprotein absorption (Kd = 0.8 PM) (28). These observations raise several questions about the properties of sulfite metabolism and the causes of its toxicity in animals, for example, whether or not sulfite oxidation in rho proceeds exclusively through the interaction of sulfite oxidase with the respiratory chain; what is the effect of sulfite metabolism on cellular energy production and other cellular functions; whether or not the radical chain reaction of sulfite oxidation occurs in vivo; and whether or not the inhibitory action of sulfite on various enzymes is observable in ho. With these points in mind, attempts were made in the present study to examine the properties of sulfite oxidation in the hemoglobin-free perfused rat liver as well as in isolated mitochondrial fractions. EXPERIMENTAL

PROCEDURES

Materials. Standard chemicals were obtained either from Fisher Scientific Company (Pittsburgh, PA) or Baker Chemical Company (Phillipsburg, NJ). Hexokinase, ADP, scopoletin, and G-6-P4 were obtained from Sigma Chemical Company (St. Louis, MO). G-6-P dehydrogenase and lactate dehydrogenase were purchased from Boehringer Mannheim Company (New York, NY). Rosaniline was obtained from K and K Laboratories, Inc. (Plainview, NY). The normal protein test diet used in tungsten-pretreatment of the rats was obtained from Nutritional Biochemicals Co. (Cleveland, OH). Assay of sulfite oxidation with isolated mitochondriul fractions. After perfusion of rat liver with cold 0.9% NaCl solution, a 10% homogenate was prepared in 0.225 M mannitol, 0.075 M sucrose, 0.1 mM EDTA, and 10 mM Tris-HCl buffer, pH 7.4. The 4 Abbreviations used: G-6-P, glucose-bphosphate; and G-6-Pase, glucose-6-phosphatase; PN, pyridine nucleotides; Fp, flavoprotein; SDH, succinate dehydrogenase; DHase, dehydrogenases coupled with NADP+; GSH, glutathione; and GSSG, oxidized glutathione.

516

OSHINO

AND

mitochondrial fraction was obtained by serial centrifugations of the homogenate at 5OOg for 10 min. The supernatant fraction and fluffy layer were discarded and the pellet thus obtained was washed once with the above-mentioned medium, and used as the mitochondrial fraction. The reaction mixture for sulfite-dependent ATP formation consisted of 0.225 M mannitol, 0.075 M sucrose, 30 mM ethanol, 10 mM potassium phosphate buffer, pH 7.4, with 10 mM glucose, 1 FM antimycin A, 2 PM rotenone, 4 III/ml of hexokinase, 0.4 mM ADP, and an appropriate amount of mitochondrial protein (1.5-5 ml/ml). Sodium bisulfite was dissolved daily in 0.1 M mannitol and the pH of the solution was adjusted to pH 7.4 by NaOH. The concentration of the sulfite solution for the calculation of the experimental results was based upon the weight of the added bisultite and hence there may be a slight overestimation of the sulfite concentration because of the slow auto-oxidation of sulfite. The reaction was initiated by addition of sulfite, and incubation was continued at 30°C for O-15 min with constant shaking. The reaction was terminated by the addition of cold perchloric acid solution at a final concentration of 3.5%, and after deproteinization the G-6-P concentration was measured using the G-6-P dehydrogenase reaction. When succinate (4 rnhrl was used as a respiratory substrate, antimycin A was omitted from the reaction mixture. Oxygen consumption was measured with a Clarktype oxygen electrode in the same incubation medium at 3O”C, but some additions, for example, the ATP-trapping system, were omitted, depending upon the purpose of the experiment. Sulfite oxidation in perfused liver. Male rats of the Sprague-Dawley strain, weighing 180 to 250 g and fed on a commercial diet, were used. Tungstenpretreatment was performed according to the procedure reported by Cohen et al. (16). Sulfite-cytochrome c reductase activity was determined in the liver of control and tungsten-pretreated rats under the conditions of Wattiaux and Wattiaux (3). The perfusion procedure and experimental conditions have been described elsewhere (29-31). The perfusion medium consisted of 115 mM NaCl, 5.9 mM KCl, 1.2 mM NaH*PO,, 1.2 mM MgCl*, 2.5 mM CaCl, 1.2 mM Na$SOa, and 25 mM NaHCO,. When desired, 10 mM glucose or 1 mM lactate and 0.3 mM pyruvate were added. The flow rate was 28-32 ml/min, and the temperature of the liver surface was 30” + 2°C. The time-sharing dual wavelength spectrophotometer and fluorometer (29) was used for simultaneous spectrophotometric measurements of the redox state of cytochrome c at 550-540 nm and of the steady state concentration of the catalase-HP, intermediate at 660-640 nm, and fluorometric measurements of the redox states of pyridine nucleotides (excitation at 366 nm, emission at 460 nm) and flavoproteins (excitation at 460 nm, emission at 540 nm).

CHANCE The O2 consumption in the liver was calculated from the effluent 0, concentration, monitored by a Clarktype oxygen electrode inserted into the tubing system just after the liver. The sulfite solution, dissolved in water and adjusted to pH 7.4, was infused into the liver through a joint in the tubing. The concentration of sulfite in the perfusate was changed either in a stepwise fashion or as a continuous linear concentration gradient from 0 to 1.7 mM. In order to prevent possible sullite auto-oxidation during the assay, sodium tetramercury chloride at a final concentration of 5 mM was infused into the stream of effluent perfusate and the perfusate was collected for a one minute interval. An aliquot of the perfusate thus collected (lo-40 ~1) was immediately transferred into a test tube containing 2 ml of a solution of 0.004% bleached rosaniline and 0.02% formaldehyde. After 30 min, the sulfite concentration in the test tube was determined at 560 nm as described by West and Gaeke (32). This measurement was also used to determine the infused sulfite concentration in each experiment. A dual-wavelength scanning spectrophotometer was used for measuring the difference spectra of the perfused liver, as reported previously (33). Other methods. Hz02 production in the mitochondrial fraction was measured by the scopoletin/horseradish peroxidase reaction (34). The rate of H,O, production in the perfused liver was calculated from the susceptibility of the steady state of the catalaseH20, intermediate to methanol, as described previously (31). Pigeon heart mitochondria were prepared by the method of Chance and Hagihara (35). Protein was determined by the biuret reaction using crystalline bovine serum albumin as a standard (36). RESULTS

Isolated

Mitochondrial

Systems

Factors affecting sulfite oxidation in isolated mitochondria. Some properties of sulfite oxidation catalyzed by rat liver mitochondria are illustrated in Fig. 1. Trace A indicates the variations in mitochondrial O2 consumption caused by different concentrations of sulfite in the presence of antimytin A. The sharp breaking-point in this trace shows that the reaction proceeds linearly until almost all the added sulfite is oxidized. In Trace B in the presence of added ADP and inorganic phosphate, the rate of O2 utilization by added sulfite increases almost twofold in this particular preparation; the stimulator-y effect of ADP was found to be somewhat variable, depending upon the mitochondrial preparation. In the sulfite concentration range

SULFITE

OXIDATION

IN PERFUSED

LIVER

517

FIG. 1. SOz2--dependent O2 consumption by rat liver and pigeon heart mitochondria. The reaction mixture contained 0.075 Msucrose, 0.227 M mannitol, 30 mM ethanol, and 10 mhf potassium phosphate buffer, pH 7.4. O2 consumption was measured by a Clark-type 0, electrode at 30°C. Antimycin A (2 PM), ADP (0.5 mM), and various concentrations of sulfite were added as indicated in the figure. Protein concentrations of rat liver and pigeon heart mitochondria were 2.9 mg/ml (A, B, and C) and 3.0 mg/ml (D), respectively.

from 25 to 500 PM, the mean value of the ratio of sulfite added to O2 consumed (S03VO) is 0.9 mol/atm. In Trace C, the addition of cyanide, an inhibitor of cytochrome oxidase, slows the rate of sulfiteinduced oxygen consumption; furthermore, in the presence of cyanide the rate of 0, utilization is more severely affected by the sulfite concentration, and the SO32-/0 ratio decreases from 0.8 with 25 PM sulfite to 0.4 with 400 PM sulfite under these conditions. In cornpal 3on, Trace D shows that pigeon heart mitochondria prepared by the protease-pretreated method (35) do not show any stimulation of 0% consumption by added sulfite at concentrations as high as 5.5 mM. Although heart mitochondria may contain sulfite oxidase (61, the enzyme is very sensitive to protease pretreatment and thus the activity may be lost in this method of preparation. The result, however, confirms that neither auto-oxida-

tion of sulfite by molecular oxygen nor direct reaction of sulfite with mitochondrial respiratory components occurs under these conditions. It should be noted that the reaction mixture contains 0.225 M mannitol, sufficient to prevent the radical chain reaction of sulfite auto-oxidation (18). In Fig. 2, the effect of O2 concentration on sulfite oxidation is compared in the presence and absence of cyanide. In the absence of cyanide, antimycin A-insensitive respiration is greatly diminished by decreasing O2 concentrations ranging from 230 to 120 PM 0,; when a correction is made for this effect, it can be seen that the rates of sulfite oxidation by the mitochondrial respiratory chain are identical at O2 concentrations of 220-160 /AM 120-70 pM, and 50-15 PM (Trace A). In the presence of cyanide, however, Trace B shows that the rate of sulfite oxidation decreases significantly as the O2 concentration decreases;

518

OSHINO

AND

FIG. 2. Effect of 0, concentrations on the SO,*-dependent Oz consumption in the absence and presence of cyanide. The reaction conditions were as described for Fig. 1, except that the reaction mixture initially contained 4 mM succinate. When 0, concentration in the medium was decreased to a desired concentration by mitochondrial respiration, antimytin A (2 PM) and ADP (0.3 mr.r) (A) or KCN (0.5 mM) (B) were added subsequently, and the reaction was started by addition of 1 mM sulfite. Mitochondrial protein concentration was 3.2 mg/ml.

at 115 PM, it is 63% of that at 230 PM, and at 45 PM O2 it is only 27%. ATP formation associated with sulfite oxidation. Figure 3 shows the time course of ATP formation associated with the oxidation of 1 mM sulfite in the presence of antimycin A. In Fig. 3A, the preparation of rat liver mitochondria exhibits a sulfitedependent O2 consumption of 77 pM/min, and in agreement with this rate of O2 utilization, the concentration of ATP measured by G-6-P formation reaches a maximum after 10 min of incubation and then slowly declines, probably due to the decomposition of G-6-P by G-6-Pase. In the experiment shown in Fig. 3B, the rate of sulfitedependent O2 consumption was 40 pM/min and the linear production of ATP continues during the 15-min incubation period at this lower rate. As a control, succinatedependent ATP formation was measured under similar conditions with respect to the rate of O2 consumption. The P/O ratio obtained with succinate was 0.77 in A and 0.52 in B, whereas the P/O ratio with sulfite was 0.41 and 0.23, respectively. Although the recovery of ATP as G-6-P was

CHANCE

rather poor under our experimental conditions, it is apparent that the P/O ratio with sulfite corresponds to half that observed with succinate in each preparation; hence, an involvement of one phosphorylating site in the sulfite oxidation system is considered. Ha2 production during sulfite oxidation in mitochondria. Hz02 production during the mitochondrial oxidation of sulfite examined using the scopolewas tin/horseradish peroxidase method (34). No detectable increase in HzOz production was seen upon addition of sulfite. This result is confirmed by the observation that the rate of sulfite-dependent O2 utilization was the same in the presence and in the absence of 30 mM ethanol, which is a hydrogen donor for catalase and stimulates the peroxidatic decomposition of H,Oz by this

2 ““1 / z L?

/

0.4

I

i

“;tE k 0

510150

5TrET

Trne

(min)

FIG. 3. Time courses of the S032--dependent phosphorylation of ADP by mitochondria. The reaction mixture contained 9.225 M mannitol, 9.975 M sucrose, 1 PM antimycin A, 2 PM rotenone, 10 mbf potassium phosphate buffer, pH 7.4, 10 mM glucose, 4 III/ml of hexokinase, 0.3 mM ADP, 30 mM ethanol and mitochondria. The reaction was initiated by addition of sulfite (1 mM) or succinate (4 mM) and incubation was continued at 30°C for O-15 min with constant shaking. When succinate was used as substrate, antimycin A was omitted from the medium. Each value represents the mean value of three separate measurements. (A) Mitochondrial protein concentration of 4.9 mg/ml and 0, consumption of 77 PMjmin with sulfite c-0-1, mitochondrial concentration of 1.5 mg/ml and 0, consumption of 150 &min with succinate (-0-l. (B) Mitochondrial concentration of 3.0 mg/ml and 0, consumption of 40 pM/min with sulfite c-O-1, mitochondrial concentration of 1.2 mg/ml and 0, consumption of 87 pM/min with succinate (-0-I.

SULFITE

OXIDATION

enzyme (37). Because no suitable method for measuring HIOz production is applicable in the presence of cyanide, there is no direct evidence for the concomitant production of HzOz in the cyanide-insensitive oxidation of sulfite in mitochondria. However, the observation that progressive inhibition of the respiratory chain by cyanide causes a corresponding decrease in the SOz2-/0 ratio towards 0.5 (cf. Fig. 10 can be taken to indicate H202 production in the direct reaction of sulfite oxidase with molecular oxygen (Eq. [31). These results with isolated mitochondria indicate, in agreement with the work

IN

PERFUSED

519

LIVER

of Cohen et al. (5), that sulfite oxidation proceeds exclusively via the respiratory chain, and thereby produces 1 mol of ATP per mol of sulfite oxidized. In comparison with the cyanide-insensitive sulfite oxidation, the oxidation of sulfite via the respiratory chain is characterized by (1) its low K, of O2 and SOs2-; (2) the SOs2-/0 ratio of 1; (3) the ATP/O (ATP/S032-) ratio of 1. The Perfused

Liver System

Difference spectra of perfused liver produced by sulfite infusion. Figure 4 shows optical difference spectra of perfused liver in various experimental conditions. In

A

m

h lnm)

4. Difference spectra of perfused liver under various conditions. The liver of the fed rat was perfused with the saline-bicarbonate solution saturated with 95% Oz-5% CO, at 30°C. The flow rate of perfusion was 30 ml/min. (A) difference spectrum of anaerobic minus aerobic liver, (Bl antimycin A-induced difference spectrum under aerobic conditions, (C) S0,2--induced difference spectra in the presence of antimycin A, (D) cyanide (25 /,&-induced difference spectrum in the presence of antimycin A, (El SOS*--induced difference spectra in the presence of antimycin A and cyanide. FIG.

520

OSHINO

AND

Row A, the anaerobic minus aerobic difference spectrum of perfused liver shows reduction of cytochrome b, c, and a + a3 with absorbance maxima at 562, 550, and 607 nm, respectively. After reoxygenation of the liver, antimycin A was infused which, as shown in Row B, results in the reduction of the b-type cytochromes around 562 nm and a slight oxidation of cytochrome c at 550 nm. This spectrum was recorded as a flat baseline in Row C, where infusion of sulfite in the presence of antimycin A is seen to cause very little reduction of cytochrome c; the slight reduction of the b-type cytochromes in the region of 560 nm cannot be assigned to a b-type heme of sulfite oxidase (6) since the concentration of this pigment is too low to be identified in the spectrum. In Row D, after a recovery of the redox state to the initial condition following cessation of sulfite infusion, the infusion of 25 PM KCN causes only a small inhibition of cytochrome a + a3 and partial reduction of cytochrome c. The absorption change above 600 nm is a consequence of the formation of a catalaselcytidine nucleotide complex (30). The spectrum of Row D was then recorded as a flat baseline for that of Row E, where the infusion of sulfite under these conditions causes a clear reduction of cytochrome c and cytochrome a + a3 even with 0.5 mM sulfite. Since the system contains antimycin A, which inhibits the respiratory chain between cytochrome b and cytochrome c, it is apparent that reducing equivalents are transferred from sulfite to cytochrome c in the respiratory chain via sulfite oxidase. Redox changes associated with sulfite oxidation in perfused liver. The effects of sulfite infusion on the redox states of pyridine nucleotides, flavoproteins and cytochrome c, on the steady state concentration of the catalase-H,Oz intermediate (Compound I), and on the rate of O2 consumption are measured simultaneously in Fig. 5. In this experiment, the concentration of sulfite infused was increased linearly from 0 to 1.45 mM. As the sulfite concentration increases, not only does the rate of O2 utilization increase, but also the redox states of the respiratory components are shifted in the direction of reduction. When the concentration of infused sul-

CHANCE Sulfur Infused (mM) b Y

-g 5

5%

100

hear

Concentration

145

N,

24

28

02

Gradlent

I0

03

E$O2 5 ,j 02

01 o 0

4

8

12 16 Tlmehn)

20

32

FIG. 5. Effects of SO,*on the redox states of pyridine nucleotides, flavoproteins and cytochrome c, on the steady state concentration of catalase-HzOz and on the effluent Oz concentration. Relative reduction states of pyridine nucleotides (PN) and flavoproteins (Fpl were measured by means of surface fluorimetry at 460 nm and 540 nm with excitation at 366 nm and 460 nm, respectively. Cytochrome c and catalase-H,Ot were measured spectrophotometritally at 550-540 nm and 660-640 nm, respectively, through a liver lobe. The flow rate of perfusate was 32 ml/min and the liver used was 12.0 g wet wt. Sulfite concentration was increased linearly from 0 to 1.45 mM as illustrated in Fig. 9. Relative reduction state of each component was expressed, assuming aerobic steady state as 0% and anaerobic steady state as 100%.

fite is changed in a stepwise fashion as in Fig. 6A, corresponding changes in the redox states of these components were also observed. Ethanol infusion at 2 mM is known to produce a highly reduced state of cytoplasmic NAD+ (38). In Fig. 6B, the reduction of pyridine nucleotides caused by ethanol and by sulfite are very nearly additive, but their effects on mitochondrial flavoproteins and cytochrome c are less in accordiance with cushioning effect of the respiratory carrier pools. Effect of sulfite oxidation on Ha, pro-

SULFITE MeOH 5mM

OXIDATION

5 ,*5, I ,05,cJq Sulfite(mM1

PERFUSED

LIVER

521

NZ

v-

_,

IN

AA=002 T

Lo

B

FIG. 6. Effects of ethanol on the sulfite-induced changes in the redox states of pyridine nucleotides and flavoproteins. The perfusion conditions were similar to those described for Fig. 5, except that sulfite concentrations were changed in a step wise fashion in the presence of 5 mM methanol (A) or 2 mM ethanol (B).

duction in perfused liver. As reported previously (37, 39), the steady state concentration of the catalase-H,Oz intermediate is a sensitive indicator for changes in the rate of H,Oz production in the liver. If HzOz production were to increase as a result of sulfite oxidation, there would be a concomitant increase in the steady state concentration of the catalase-H,Oz intermediate. However, as seen in Row D of Fig. 5, higher concentrations of sulfite cause a slight decrease (~10%) rather than an increase in concentration of the catalaseH202 intermediate (AA,,,,, .,). Assuming this decrease at 660-640 nm to be due solely to the catalase-H,O, intermediate, it would indicate a slight decrease in the rate of Hz02 production (31); however, a similar decrease at 660-640 nm is observed in the presence of ethanol or methanol, where the steady state concentration of the catalase-H,O, intermediate is already zero (Fig. 6). Therefore, the decrease in absorbance at 660-640 nm observed on infusion of sulfite may be ascribed to an unknown chromophore absorbing at this

wavelength pair. The results of Figs. 5 and 6 indicate that, in the perfused liver, sulfite oxidase does not react directly with molecular oxygen under normal conditions, i.e., there is no detectable increase in H,Oz generation; instead, the oxidation of sulfite takes place exclusively through the mitochondrial respiratory chain, as indicated in Eqs. [ll and 121. Additional support for this conclusion is afforded by the observation that the rate of O2 consumption stimulated by sulfite is identical in the presence and absence of 5 mM methanol, ascertained in separate experiments (not shown). In the presence of 5 mM methanol in the perfused liver, catalase decomposes H,O, by a peroxidatic mechanism, i.e., H,Oz + methanol -+ 2H20 + formaldehyde, whereas in the absence of a hydrogen donor such as methanol, catalase functions in its catalatic mode, i.e., 2H,02 -+ 2H,O + O2 (31). Stoichiometry of sulfite oxidation and oxygen consumption in perfused liver. In order to measure the rate of sulfite oxidation in the perfused liver, the sulfite con-

522

OSHINO

AND

centration in the influent perfusate was increased linearly and the concentration difference between the influent and effluent perfusates was determined (a representative result is shown in Fig. 9). In Fig. 7A, the rate of sulfite oxidation thus measured is shown as a function of the rate of sulfite infusion. Figure 7B shows the rate of sulfite-dependent O2 consumption, also measured from the concentration difference in the perfusate with and without sulfite. These data characterized sulfite oxidation in perfused liver as follows: (1) Both the rate of sulfite oxidation and that of oxygen consumption are increased almost linearly up to 3 pm01 of sulfite infused/min per g wet wt of liver, indicating that the sulfite oxidation reaction is not saturated even with sulfite concentration of more than 1 mM in the perfusate; (2) regardless of the rate of sulfite infusion, between 25 and 40% of the sulfite infused is removed from the perfusate under these conditions; (3) the ratio of sulfite oxidized to O2 consumed was between 1.6 and 1.4 mol/atm with infusion rates below 3 pmol/min/g wet wt of liver at 30°C. Sulfite oxidation and associated Ha2 production in the presence of cyanide. As in the case of isolated mitochondria, sulfite oxidation in the perfused liver appears to

CHANCE

proceed via the respiratory chain, so that complete inhibition of the respiratory chain by cyanide should elicit a direct reaction of sulfite oxidase with molecular oxygen. The traces of Fig. 8 are representative of measurements of O2 consumption in the effluent perfusate and of the redox state of pyridine nucleotides in the liver. Upon infusion of 0.5 mM KCN, the pyridine nucleotides become more reduced and the rate of O2 consumption is inhibited by 75%. Addition of sulfite in the presence of cyanide now causes an oxygen consumption twofold greater than that observed without cyanide. However, the rate of sulfite oxidation is similar in the presence and absence of 0.5 mM cyanide (Table I) and thus the S032-/0 ratio observed in the presence of cyanide is half that observed without cyanide (0.6-0.8). Cyanide causes not only an inhibition of respiratory chain but also the complete conversion of catalase into the inactive catalase-cytidine nucleotide complex (30). Under these conditions, the H202 produced in sulfite oxidation may react with glutathione-peroxidase to cause oxidation of NADPH, as observed with t-butylperoxide infusion (40, 41). Figure 8 indicates this to be the case in perfused liver; in contrast to the reduction of pyridine nucleotides obIB

Sulfite Infused lpmoles/m~n/g

)

FIG. 7. The rates of SOz2- oxidation and of O2 consumption in the liver from the control and tungsten-pretreated rats. The experiments (three for each group of animals) were performed as described for Figs. 5 and 9. The rate of sulfite oxidation was calculated from the concentration difference between the influent and effluent SO,‘- concentration (A). The rate of O2 consumption were also calculated from the concentration difference in the effluent perfusate with and without sulfite infusion (B). (-0-j: liver of control rats, (-0-j: liver of the rat pretreated with 100 ppm tungsten for 8 days.

SULFITE

01

,

2U

OXIDATION

I

24

28

32 36 40 44 Perfwon Tbme hn)

48

52

FIG. 8. Effects of cyanide-insensitive SOa2- oxidation on the redox state of pyridine nucleotides and on the O2 consumption. The experimental conditions were as described for Fig. 6. KCN (pH 8) was infused in a final concentration of 0.5 mM as indicated in the figure.

served in the absence of cyanide, sulfite infusion in the presence of cyanide causes a slow but remarkable oxidation of pyridine nucleotides. Simultaneous measurement of the redox state of cytochrome c (not shown) confirmed the lack of oxidation of the respiratory components under these conditions, indicating that the observed oxidation of pyridine nucleotides could be attributed to the oxidation of NADPH through the enhanced reaction of GSH-peroxidase. The extent and time course of the pyridine nucleotide oxidation indicated by the fluorescence change are similar to those of the oxidation seen on infusion of t-butylperoxide at a rate corresponding to the rate of H,Oz production induced by sulfite oxidation under these conditions (N. Oshino, unpublished observation). Sulfite oxidation in the perfused liver of tungsten-pretreated rats. Pretreatment of rats with tungsten causes a decrease in the hepatic content of active sulfite oxidase, with a half-time of 4.7 days (16). Therefore, rats were maintained for 8 days on a low molybdenum diet with drinking water supplemented with 100 ppm of tungsten. In agreement with the results reported by Cohen et al. (16), sulfite oxidase activity in the liver of the pretreated rats was 23% of the control value. Figure 9 shows repre-

IN

PERFUSED

LIVER

523

sentative experimental traces obtained from the liver of a tungsten-pretreated rat; the traces of Fig. 9A show the sulfite concentrations in the influent and effluent per&sates, and those of Fig. 9B and 9C represent, respectively, the changes in the redox state of pyridine nucleotides and in the O2concentration in the effluent perfusate. The rates of sulfate oxidation and O2 consumption obtained in three separate experiments have been given in Fig. 7A and 7B, respectively. These two figures characterize the oxidation of sulfite in the perfused liver of tungsten-pretreated rats as follows: (1) low concentrations of sulfite cause reduction of pyridine nucleotides as in the control liver but after this initial reduction, a return to the initial level is seen as the sulfite concentration increases; upon termination of sulfite infusion, therefore, no reoxidation of pyridine nucleotides is observed. This phenomenon is in contrast to the clear oxidation observed in the control liver on termination of sulfite infu-

FIG. 9. Redox change of pyridine nucleotides caused by continuous increase of SO,‘concentration in the liver of tungsten-pretreated rat. The perfusion conditions were as described for Fig. 5, except that linear gradient of SO,*- concentration infused was from 0 to 1.6 mM. Row A: SO,*- concentration in the influent (-0-j and effluent (-0-j perfusates. Row B: relative reduction state of pyridine nucleotides. Row C: 0, concentration in the etIluent perfusate.

524

OSHINO

AND

sion, as shown in Fig. 5. (2) At low sulfite concentrations, the rates of sulfite oxidation are similar in the control and tungsten-pretreated rats, but in the latter a maximal rate is achieved with sulfite infusion rates above 1.5 pmol/min/g wet wt of liver. (3) In contrast to the rate of sulfite oxidation, the rate of O2 consumption in the liver of the pretreated rat is between 52 and 60% of that in the control at infusion rates below 1.5 pmol/min/g wet wt of liver. (4) The S032-/0 ratio is as high as 2.1 in the tungsten-pretreated liver at infusion rates below 1 pmol/min/g wet wt of liver, and appears to decrease to 1.2 with faster rates of sulfite infusion. DISCUSSION

The present study confirms the results obtained with purified sulfite oxidase (6, 7) and with isolated mitochondria (5) and extends them to the investigation of the properties of the sulfite oxidation reaction in perfused liver. It has been reported that the apparent K, values for sulfite and cytochrome c in the sulfite-cytochrome c reductase activity of purified sulfite oxidase are 12 and 0.3 PM, respectively (6). The sulfiteO2 reductase activity of sulfite oxidase seems to have rather high K, values, since the reported values of K, for sulfite and 0, are 140 and 580 PM, respectively (7). In agreement with these data, the direct reaction of sulfite oxidase with 02, which is characterized by its low affinity for O2 and also for sulfite (Figs. 1 and 21, could not be detected in these studies unless the respiratory chain was heavily inhibited by cyanide. The S03*-/O ratio was determined to be 0.9 which, within the experimental error, confirmed the conversion of O2 to H20 (Eqs. 111 and [21), but not to H202 (Eq. [31), and indicated that sulfite oxidation proceeds exclusively through the interaction of sulfite oxidase with cytochrome c in the respiratory chain under normal physiological conditions. Probably because of the rather slow rate of sulfite-dependent respiration under our experimental conditions (in mannitol/sucrose/phosphate medium), ATP formation measured as G-6-P during sulfite oxidation was only 0.2-0.4 per 0, atm. As

CHANCE

reported by Slater et al. (42), the phosphate potential in mitochondria may not lead to a sufficiently high ATP level to counteract the rate of endogenous ATP utilization. Thus when the respiratory rate is relatively slow, the conditions may not be suitable for the detection of ATP formation. This may account for the low recovery of ATP as G-6-P under our experimental conditions. A further cause for our low values may be the existence of G-6-Pase activity, as indicated by the slow decline of G-6-P concentration after the consumption of added sulfite (Fig. 3). However, the ATP/O ratio observed with succinate under similar conditions was 0.5-0.8; thus the ATP/O ratio obtained with sulfite is half that found with succinate. Since succinate-dependent respiration has been established to include two phosphorylation sites, it is reasonable to conclude from these results that the reducing equivalent from sulfite enters the respiratory chain via cytochrome c and is utilized in the phosphorylation reaction at Site III (see Fig. 10). Of interest is the hypothesis that sulfite oxidase is located in the inter-membrane space of the mitochondrion (3-5). In fact, the results of this paper demonstrate that the interaction of sulfite oxidase with cytochrome c, which is located on the outer side of the inner mitochondrial membrane

d

m

jslte

i

FIG. 10. Scheme for possible interaction between sulfite oxidase and mitochondrial components. Fp,: NADH-cytochrome b, reductase, Fp: NADH-CoQ reductase, SDH: succinate dehydrogenase, DHase: dehydrogenases coupled with NAD+, Q: coenzyme Q, SO: sulfite oxidase.

SULFITE

OXIDATION

IN

(43, 44), is so effective that the direct reaction of sulfite oxidase with O2 does not occur in suspensions of isolated mitochondria. It should be noted that NADH-cytochrome b, reductase and cytochrome b, are located in the outer mitochondrial membrane (45, 46). This enzyme system, in the isolated state, exhibits cytochrome c reductase activity and yet cannot supply reducing equivalents from NADH to cytochrome c in intact mitochondria (45, 47). Thus, the different capabilities of sulfite oxidase and NADH-cytochrome b5 reductase for reducing mitochondrial cytochrome c may be a consequence of the different localization of these enzymes in mitochondrial compartments. Fukushima et al. (46) reported that isolated sulfite oxidase could interact with the NADH-cytochrome b, reductase system and exhibit slow cytochrome c reductase activity. There is a similar observation that the molybdenum signal of sulfite oxidase in rat liver mitochondria can be slowly reduced by NADH, probably through some flavoproteins (5). Thus, sulfite oxidase may function as a transfer mechanism for reducing equivalents from the cytosolic to the mitochondrial space. However, no significant effect of an altered NADH level induced by infusion of ethanol or by a change in the lactatelpyruvate ratio has as yet been detected on sulfite oxidation in the perfused liver. In the perfused liver, all lines of evidence obtained in the present study point to preferential transfer of reducing equivaTHE RATES OF SULFITE Expt.

no.

Conditions

1

Aerobic + KCN

0.5 mru

2

Aerobic + KCN

0.5 mM

Aerobic + KCN

0.5 mM

3

OXIDATION

o The experimental conditions were intervals and the sulfite concentration value presented as SO,*- concentration was established.

PERFUSED

525

LIVER

lents from sulfite to the mitochondrial respiratory chain. The slight stimulation (~10%) of sulfite-dependent O2 consumption by antimycin A suggests that the site of interaction of sulfite oxidase with the respiratory chain is at cytochrome c in perfused liver, as it is in isolated mitochondria. The S032-/0 ratio of 1.6-1.4 in the perfused liver is rather high compared with that in the mitochondrial fraction (0.9) and is decreased to 0.8-0.6 in the presence of 0.5 mru cyanide (Table I). These high values in the SO,*-/0 ratio may be attributable to the partial replacement of endogenous respiration by sulfitedependent respiration and to the consequent underestimation in the measurement of O2 consumption. No detectable increase in the production of HzOz by sulfite oxidation was seen in the absence of cyanide, and it is concluded that under normal physiological conditions sulfite oxidation proceeds via the mitochondrial respiratory chain. Only in the presence of excess cyanide, resulting in almost complete inhibition of mitochondrial respiration, is the direct reaction of sulfite oxidase with molecular oxygen observed. Under such conditions, the production of HzOz was demonstrated by the oxidation of pyridine nucleotides, due to the acceleration of the GSHperoxidase reaction and the consequent oxidation of NADPH through NADPHGSSG reductase. It is well known that the radical chain reaction of sulfite auto-oxidation (Eqs. [491) is initiated in vitro by 02- (17-22) and

TABLE I AND OF 0, CONSUMPTION

IN PERFUSED 0, consumed (PM)

LIVERY

S03P;~;used

SOsz;;zldized

765

248 188

135 76

E

793

259 272

94 162

1.4 0.8

1480

361 278

120 378

1.5 0.7

so,2-/o (mohatm)

as described in Fig. 8. The effluent perfusate was collected at 0.5-min was determined as described in Experimental Procedures. Each is a mean value of 10 samples which were collected after steady state

526

OSHINO

AND

that the chain length of the reaction approaches 300-350 mol/mol of 02- in the chloroplast under illumination, even though this fraction contains superoxide dismutase and anti-oxidants (25). Mitochondria in vitro produce H202 (48, 49), most of which is attributable to the production and subsequent dismutation of 02from the respiratory components (50). As expected from the stimulatory effect of antimycin A on HzOz production (31, 49), the greater degree of reduction of the respiratory components in the presence of cyanide should result in an increased production of O,-; however, as shown in Table I, the rate of sulfite oxidation under these conditions is slower rather than faster than the rate observed without cyanide, indicating that 02--dependent oxidation of sulfite is negligible in the perfused liver, where the action of superoxide dismutase and/or the presence of an anti-oxidant may prevent the initiation of the chain reaction of sulfite oxidation. A steep O2 concentration gradient near the mitochondrial compartment in perfused liver (39) would be favorable for preventing this auto-oxidation reaction; furthermore, the low st&adystate concentration of sulfite in the cell under diffusion limited conditions (see below) could diminish the possible propagation of the chain reaction in Go. The rate of sulfite oxidation in perfused liver increases linearly with increasing sulfite concentration, up to 2 mM in the perfusate, and does not reach saturation even though the sulfite concentration in the effluent perfusate is only 25-40% less than the influent concentration. In spite of the great decrease (77%) in hepatic sulfite oxidase activity in the tungsten-pretreated rat, sulfite oxidation under these conditions approaches saturation only when the sulfite concentration exceeded 0.6 mM (or the infusion rate exceeded 1.5 pmol/min per g wet wt of liver; cf. Fig. 7). These observations are in sharp contrast with the low K, value (