Thiosulfate reductase from Chlamydomonas

........,.... • ••••• AL.F •

© 1997 by Gustav Fischer Verlag, Jena

Thiosulfate reductase from Chlamydomonas JOSE L. PRIETO, JOSE R. PEREZ-CASTINEIRA*, and JOSE M. VEGA Instituto de Bioqufmica Vegetal y Fotosfntesis, Centro de Investigaciones Ciendficas Isla de la Canuja, Universidad de Sevilla y CSIC, 41092-Sevilla, Spain Received December 13, 1996 . Accepted February 26,1997

Summary

Thiosulfate reductase (thiosulfate-thiol sulfunransferase, (EC 2.8.1.3) TSR) and rhodanese (thiosulfatecyanide sulfurtransferase, (EC 2.8.1.1) RDN) activities have been identified in crude extracts from the eukaryotic green alga Chlamydomonas reinhardtii. These activities co-purified after several purification steps and no isoforms have been separated. Kinetic studies indicate that dithiols (DTE) exert a competitive inhibition on rhodanese activity, the affinity for DTE being 28-fold higher than that for cyanide. These results support the idea that both activities are mediated by the same protein. The activities are not sensitive to thiol reagents, whereas they are inhibited by specific arginine-modifying reagents. The physiological role of this enzyme could be basically the assimilation of S-sulfane by the alga.

Key words: Chlamydomonas reinhardtii, rhodanese, sulfur metabolism, sulfurtransferase, thiosulfate reductase.

Abbreviations: A = absorbance; Chl = chlorophyll; DTE = dithioerythritol; DTT = dithiothreitol; pHMB =p-hydroxymercuribenzoate; PLP = pyridoxal-5'-phosphate; RDN = rodanese; TNBS = trinitrobenzensulfonic acid; TSR =thiosulfate reductase. Introduction

Thiosulfate has been identified as the major acid-volatile product formed when extracts obtained from different organisms such as S. cerevisiae, C reinhardtii and others are prepared in the presence of ATp, and a reductant (thiols or reduced pyridine nucleotide) (Hodson and Schiff, 1971). Fungi (Chauncey and Wesdey, 1983; Thomas et aI., 1992; Uhteg and Wesdey, 1979) and photosynthetic organisms such as phototrophic bacteria (Truper and Pfennig, 1966), cyanobacteria (Schmidt et aI., 1982), green algae (Hodson et aI., 1968) and higher-plant tissue cultures (Hart and Filner, 1969) can grow using thiosulfate as the only sulfur source. Biochemical pathways where thiosulfate might be involved have been reported in bacteria (Hulanicka et aI., 1986; Nakamura et aI., 1983, 1984) and fungi (Kitano et aI., 1985; Lydiate et aI., 1988; Thomas et aI., 1992); however, litde information is available for photosynthetic organisms.

Mi+

* Correspondence. j. Plant Physiol. WJL 151. pp. 385-389 (1997)

Thiosulfate: acceptor sulfurtransferases mediate the transference of a sulfur atom from sulfane-sulfur containing compounds to a thiophilic acceptor. Donors include thiosulfate, organic thiosulfonates (R-S(Oz)S-) and persulfides (R-(SJS-). Acceptors can also be variable and, depending on their nature, the activity of these enzymes have different names: when the acceptor is cyanide, the activity is known as «rhodanese,., whereas if the acceptor is a thiol the activity is called «thiosulfate reductase» (Wesdey, 1981).

solsol-

S20/- + CN- ~ SCN- + (rhodanese) S20/- + RSH ~ RSSH + (thiosulfate reductase)

Although all thiosulfate: acceptor sulfurtransferases mediate both reactions, some of them are preferentially rhodaneses and others thiosulfate reduccases. Rhodaneses have a higher affinity for cyanide than for thiols as the thiophilic acceptor, whereas thiosulfate reductases prefer thiols instead of cyanide. Furthermore, rhodaneses and thiosulfate reducta-

386

JOSE L. PRIETO, JOSE R. PEREZ-CAsnNEIRA, and JOSE M. VEGA

ses proceed through different catalytic mechanisms (Westley, 1981; Chauncey and Westley, 1983). The possible physiological role of these activities is a matter of controversy; thus, although traditionally rhodanese has been accepted to playa role in cyanide detoxification (Sykes, 1981), some authors have proposed that this enzyme is also involved in the synthesis of iron-sulfur dusters (Cerletti, 1986). Thiosulfate reductase might be involved in sulfur assimilation in Saccharomyces cerevisiae and Salmonella typhimurium (Hulanicka et al., 1986; Nakamura et al., 1983, 1984; Thomas et al., 1992). This enzyme has also been identified in Chiarella fusca (Schmidt et al., 1984), cyanobacteria and higher plants (Schmidt, 1986). In this paper, we report the identification, purification and characterization of a thiosulfate: acceptor sulfurtransferase in the green alga Chlamydomonas reinhardtii. This enzyme can mediate either rhodanese or thiosulfate reductase activities. Kinetic data suggest that the enzyme is a real thiosulfate reductase. Materials and Methods

Chemicals CHES, DTT, DTE, cysteine, lipoic acid and glutathione were purchased from Sigma (St. Luis, MO, USA). DEAE-sephacel and phenyl-sepharose were from Pharrnacia (Uppsala, Sweden). FolinCiocalteau reagent and salts for culture media were obtained from Merck (Darmstadt, Germany).

Cell growth and preparation ofcellfoe extracts Chlamydomonas rrinhardtii strain 21 gr cells were grown autotrophically in liquid medium with 10 mmol/L NH4CI and 0.3 mmol/L Na2S04 as nitrogen and sulfur sources respectively, as previously reported (lOOn and Vega, 1991). Cells were collected during the exponential phase of growth (~nm = 1.5-2) by centrifugation at 5,000 &. for 5 min and broken by freezing in liquid nitrogen for 90 s. Frozen cells were thawed and resuspended in working buffer (10 mmol/L CHES-KOH, pH 9.5, 5 mmol/L DTT) and the suspension was Stirred for 1 h at 4·C. Unbroken cells and debris were removed by centrifugation at 16,000 &. for 15 min at 4 ·C and the supernatant was utilized as starting material for enzyme purification. Chlorophyll and protein determination Chlorophyll was determined by the method of Arnon (1949) and protein was estimated by using Peterson's adaptation of the Lowry method (Peterson, 1977) with bovine serum albumin as standard.

Thiosulfate reductase assay Thiosulfate reductase activity was measured by monitoring sulfite production from thiosulfate using different thiols as sulfane-sulfur acceptors, according to the method of Uhteg and Westley (1979) with some modifications. The reaction mixture contained 10 mmoll L CHES-KOH, pH 9.6, 10 mmol/L Na2S203 and 5 mmol/L dithiols (DTE or DTT) or 10 mmol/L monothiols (cysteine or reduced glutathione), proceeded at 50·C for 5 min and then was stopped by adding 0.5 mL of a 0.23 M HgCh solution in water. The white precipitate formed was removed by centrifugation at 15,000 &. for 5 min. Then, 0.5 mL of the supernatant was added to a mixture

containing 1 mL of 0.02 % (v/v) formaldehyde in water and 1 mL of 0.04 % (w/v) fucsin in 0.72 mollL HCI. After 10 min absorbance was read at 600nm (eroo 3.0x 10-2 (J.1moI/L)-lcm-1).

=

Rhodanese assay Rhodanese activity was determined by measuring SCN- formation from thiosulfate and cyanide. SCN- was determined according to Sorbo (1955) as the red Fe(SCNh complex at 460 nm. The reaction mixture contained 10 mmol/L CHES-KOH, pH 9.5, 40 mmoll L KCN and 10 mmollL Na2S203' After 10 min incubation at 50 ·C, 0.25 mL of 0.5 mollL FeCl3 in 20 % H 2S04 was added to the mixture and the absorbance was read at 460 nm (£460 = 3.0 X 10- 3 (J.1mollL)-lcm-1).

Protein purification Cell extracts were obtained from about 30 g wet weight of C

reinhardtii cells as described above. Nucleic acids and pigments were

removed by adding a 2 % (w/v) protamine sulfate solution in working buffer until the final protamine concentration was 0.077% (wI v). After 10-15 min of gentle stirring at 4 ·C, the white pellet was removed by centrifugation at 27,000 &. for 15 min and the supernatant was loaded onto a DEAE-sephacel column (1 x 40 cm) equilibrated with working buffer. After thoroughly washing with working buffer, fractions containing both thiosulfate reductase and rhodanese activities were eluted with 75 mmol/L KCI in working buffer. The active fractions were pooled, concentrated by ultrafiltration and supplemented with KCI up to a final concentration of 300 mmollL. This preparation was loaded onto a phenyl-sepharose column (1 x 40 em) equilibrated with 300 mmollL KCI in working buffer. Fractions with enzyme activity were eluted with 50 mmol/L KCI in working buffer, pooled and concentrated as described above. This preparation was utilized for subsequent studies.

Results and Discussion

Both thiosulfate reductase (TSR) and rhodanese activities were detected in permeabilized cells and crude extracts (Table 1). Among thiols, the better acceptors for TSR activities were

Table 1: Characterization of thiosulfate sulfurtransferase activities from Chlamydomonas reinhardtii.

System

In vitro

Complete minus thiosulfate

100

Complete minus thiosulfate minus cyanide minus enzyme

100

In situ

TSRActivity (%)

minusDTE minus enzyme

o

0.8 1.2

100 0.1 2.2 0.5

Rhodanese activity (%) 100 o 0.1 0.3 0.1 1.1 7.5

The complete system contained in a final volume of 0.5 mL: 5 J.1moles CHES-KOH buffer, pH 9.5, 2.5 J.1moles DTE, 5 J.1moles thiosulfate and the appropriate amount of enzyme. 100 % TSR activity was 5.37U/mL (in vitro) and 0.93 U/mg chi (in situ), respectively. For rhodanese activity 20 J.1moles of cyanide were used instead ofDTE. 100% rhodanese activity was 1.9U/mL (in vitro) and 0.44 U/mg chi (in situ).

387

Thiosulf.lte reductase from Chlamydomonas

:i ~

2-

~

~

00(

0.1

0.1

0.4

0.4

0.3

0.3

I E c

0

00

0.2

0.2

0.1

0.1

200

400

800

800

('oj

1/1

,g 00(

1000

Elution Volume (mll

Fag. 1: Phenyl-sepharose chromatography of Chlamydomonas mnhardtii thiosulfate sulfurtransferase. The enzyme was adsorbed on a

phenyl-sepharose column as indicated in Materials and Methods. A stepwise elution of the protein was carried out, as indicated by the arrows, with different KCI concentrations in the working buffer. Fractions of 3 mL were collected where DTE-TSR (e) and rhodanese (0) activities were measured.

orr and

OTE, while monothiols such as cysteine, glutathione or lipoic acid were less efficient. In addition, the presence of dithiols in the working buffer was also crucial for maintaining enzyme activity along the purification procedure, while thiosulfate and ~-mercaptoethanol were less effective (data not shown). The purification of proteins capable of mediating any of the activities was then attempted utilizing the cell extracrs as starting material. The first purification step was the removal of nucleic acid and pigmenrs with protamine sulfate. The pigment and nucleic acid-free extract was then subjected to a OEAE-sephacel column; TSR and RDN activities co-eluted in the same fractions (not shown). Subsequent purification through a phenyl-sepharose column did not separate the activities that co-eluted again in the same fractions (Fig. 1). Table 2 shows a typical purification table. Both thiosulfate reductase and rhodanese activities were purified around 150fold. No isoenzymes have been detected, in contrast with the four isoenzymes reported in Chlo~l/a fosca (Schmidt et al., 1984). These resulrs suggested that thiosulfate reductase and rhodanese activities were mediated by a single sulfurtransferase in C mnhartitii. The resulting preparation showed three major bands upon native polyacrylamide gel electrophoresis, indicating that the enzyme is not in a homogeneous state (data not shown).

Optimal pH and temperature were found to be around 10 and 55·C respectively for both activities (Table 3); however, activity assays were performed at pH 9.6 and 50 ·C, in which more than 95 % activity was preserved. Moreover, both activities were remarkably heat-resistant; thus, nearly 100 % of thiosulfate reductase activity remained after 30 min incubation of the purified preparation at 70 ·C and around 60 % remained after 30 min at 90·C (Fig. 2A). However, rhodanese activity showed higher heat-sensitivity than thiosulfate reductase and only around 20 % of total activity remained after 30 min at 90·C (Fig. 2 B). In this context, it is important to indicate that during the TSR activity assay the protein is returned to near-optimal temperature (50·C) in the presence of 5 mmollL dithiol or 10 mmollL monothiol, whereas for the rhodanese assay, no thiols at all were added to the assay reaction mixture. It has been proposed that thiols protect rhodaneses against heat inactivation (Dungan and Horowitz, 1993) and, probably, they also help in protein refolding after denaturation (Horowitz and Hua, 1995); therefore it seems likely that the heat-denatured enzyme could return to irs proper conformation during the TSR assay but not during the rhodanese assay. In connection with this, it has been suggested that rhodanese can exist either in an active (reduced) or inactive (oxidized) state (Schmidt et al., 1984). Although all thiosulfate reductases can mediate rhodanese activity and viceversa, they are not identical enzymes (Westley, 1981); thus, several kinetic studies were carried out to characterize our enzyme. Kinetic data calculated using both activity assays are shown in Table 3. Km for thiosulfate was 0.5 mmollL or 1.5 mmollL for the reductase or the rhodanese

Table 3: Kinetic parameters of the thiosulfate sulfunransferase from Chlamyd6monas mnhardtii. Parameter

Enzymatic activity

pH optimum Temperature optimum (.C) E. (KJ.mol- 1) Km values (mmoUL) - Thiosulfate -DTE

DTE-TSR

Rhodanese

9.5-11.5 55 32.2

9.5-10 55 32.8 1.5

0.5 0.25 0.25 1.5 2.0

-DTT

-GSH -Cysteine -Cyanide

7.0

Table 2: Purification of thiosulfate reductase and rhodanese activities from Chlamydomonas "inhardtii. Step

Crude extract Protamine sulfate sup. DEAE-sephacel eluate Phenyl-sepharose eluate

DTE-TSR

Rhodanese

Vol (mL)

Prot (mg)

T.Act (U)

Sp.Act (U/mg)

Yield (%)

T.Act. (U)

Sp.Act. (U/mg)

Yield

140 153 278 11

569 243 9.7 0.7

164 154 81.1 30.9

0.29 0.63 9.39 44.42

100 93.6 49.4 18.8

93.7 88.4 43.3 17.6

0.165 0.364 4.48 25.26

100 94.4 46.3 18.7

The purification procedure, the standard activity assays and protein determination are described in Materials and Methods.

(%)

388

JosE L. PRIETO, JOSE R. PEREZ-CAsnNEIRA, and JosE M. VEGA

activity, respectively. As far as thiols are concerned the enzyme showed higher affinity for dithiols (OTE and OTT) than for monothiols (cysteine and glutathione). Km for cyanide (7.0 mmollL) was significantly higher than those for thiolic acceptors (Table3). The results obtained with different inhibitors also support the idea that both activities are catalysed by the same enzyme molecule (Table 4). The lack of effect on activity by sulfhydryl group blockers, such as p-hydroxymercuribenwate, methylmethanethiosulfonate (not shown) or iodoacetamide, suggests that no cysteine is essential for activity or they are buried into the protein molecule and thus unreachable by these compounds. The latter is probably the most likely explanation because previous evidence suggests that cysteine residues are essential for activity in these enzymes (Luo and Horowitz, 1994; Nagahara et al., 1995; Ploegman et al., 1978). Arginine residues did seem to be involved in enzyme activity as suggested by the inhibitory effects showed by

-

~

~

~ > ~

c(

100

Table 4: Effect of metabolites and chemical reagents on the thiosulfate reductase activity and rhodanese activity from Chlamydomonas

reinhardtii.

Inhibitor

Conc (mmoIlL)

Control pHMB pHMB Iodoacetamide TNBS PLP Phenylglyoxal

Enzymatic activity (%) DTE-TSR Rhodanese 90 min 180 min 90 min 180 min 100 103 91 98 22 75 28

0,01 0.1 1 1 5 5

100 102 91 94 12 98 32

100 119 109 65 32 89 14

100 116 102 52 27 79 21

Aliquots of purified enzyme were incubated at 4 ·C with each inhibitor in 10 mmol/L CHES-KOH, pH 9.5. At the indicated times, activities were assayed as described in Materials and Methods. 100 % TSR activity was 3.51 U/mL. 100 % rhodanese activity was 0.73U/mL.

1N

[ OTE I- 2 rnmo&tL

(mUr' 80

60

0::

en

";'

40

W l-

e

20

A 6

10

16

20

26

30

Time (min)

80

> :c u

60

Z

40

~

0.2

0.3

FIg.3: Lineweaver-Burk plot of the reaction rates versus concentration of cyanide in the presence of different concentrations of DTE. Rhodanese activity was measured as described in Materials and Methods, with different concentrations of cyanide in the presence of 2 mmollL, 5 mmol/L and 10 mmollL DTE.

c(

e

0.1

11 [eN-] (mmoIlLr'

100

~

0.0

0:: 20

B 10

16

20

26

30

Time (min)

Fig. 2: Thermal inactivation of thiosulfate reductase (A) and rhodanese (B) activities from Chlamydomonas reinhardtii. Protein samples were incubated in the assay buffer at 60·C (0), 70·C (e), 80·C (.1) and 90·C (0). At the indicated times aliquots were taken and submerged into ice; then, activity assays were performed, as indicated in Materials and Methods, by supplementing with the substrates.

TNBS and phenylglyoxal. Pyridoxal-5'-phosphate has a small effect on both activities, suggesting that lysine residues may not be essential for the enzyme activity. Site-directed mutagenesis studies performed with the ~ovine liver rhodanese have demonstrated the implication of both arginine and lysine residues in substrate binding (Luo and Horowitz, 1994; Nagahara et al., 1995; Ploegman et al., 1978). Particularly interesting are the data of Fig. 3, indicating that OTE is a competitive inhibitor (~ = 2.2 mmollL) of rhodanese activity with respect to cyanide, indicating that OTE and cyanide share the same enzyme active site. It is difficult to state now the precise physiological role for thiosulfate reductaselrhodanese activity in Chlamydomonas reinhardtii. With respect to a possible assimilatory function, the experimental evidence is contradictory; thus, algal cells can grow with thiosulfate as sulfur source L. Prieto, unpublished observations) and sulfate uptake in this organism is

a.

Thiosulfate reductase from ChLzmytbJmonas

strongly inhibited by thiosulfate under normal culture conditions (Perez.-Castifieira et al., 1992). However, thiosulfate reductase activity was not induced under sulfur-starvation conditions (not shown), which is a common characteristic of the enzymes involved in sulphur assimilation pathways (Schmidt, 1986). Moreover, no changes in enzyme activity levels were observed when cdls were subjected to different growth conditions, such as growth with different sulfur sources or with acetate as carbon source in the absence of light (data not shown). On the other hand, besides thiosulfate, the real in vivo substrate for TSR activity, other sulfane-sulfur containing compounds have been demonstrated to be substrates of Saccharomyces cerevisiae thiosulfate reductase (Chauncey and Westley, 1983). The ability of sulfur to exist in several oxidation states makes the formation of compounds such as thiosulfate and polithionates in the cells as side products of metabolic reactions possible. Thiosulfate reductase could then act as a scavenger of these compounds, recovering them back into the sulfur assimilation pathway. In any case, further biochemical and genetic studies need to be performed in order to ellucidate the physiological role of thiosulfate and thiosulfate sulfumansferases in photosynthetic organisms. Acknowledgements

This work was supponed by Research Grant n° PB93-0735 from Direcci6n General de Investigaci6n Cientffica y Tecnica (DGICYf, Spain) and Research Group CVI0118 Qunta de Andalucia, Spain).

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KITANO, K., Y. NOZAKI, and A. IMADA: Selective accumulation of unsulfated carbapenem antibiotics by sulfate transpott-negative mutants of S. Griseus subsp. Ctyophilus C-19393. Agric. BioI. Chem. 49, 677-684 (1985). LE6N, J. and J. M. VEGA: Separation and regulatoty propenies of O-acetyl-L-serine sulfhydtylase isoenzymes from ChLzmytbJmonas reinhardtii. Plant Physiol. Biochem. 29, 595-599 (1991). Luo, G-x. and P. M. HOROWITZ: The sulfunransferase activity and sttucture of rhodanese are affected by site-directed replacement of arg-186 or lys-249. J. BioI. Chem. 269, 8220-8225 (1994). LYDIATE, D. J., C. MENDEZ, H. M. KIESER, and D. A HOPWOOD: Mutation and cloning of clustered Streptomyces genes essential for sulfate metabolism. Mol. Gen. Genet. 211, 415-423 (1988). NAGAHARA, N., T. OKAZAKI, and T. NISHINO: Cytosolic mercaptopytuvate sulfunransferase is evolutionary related to mitochondrial rhodanese. J. BioI. Chem. 270, 16230-16235 (1995). NAKAMURA, T., H. IWAHASHI, and Y. EGUCHI: Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium. J. Bacteriol. 158, 1122-1127 (1984). NAKAMURA, T., Y. KON, H. IWAHASHI, and Y. EGUCHI: Evidence that thiosulfate assimilation by Salmonella typhimurium is catalysed by cysteine synthase. J. Bacteriol. 156, 656-662 (1983). PEREZ-CAsnNEIRA, J. R., J. L. PRIETO, and J. M . VEGA: Sulphate uptake in ChLzmytbJmonas reinhardtii. Phyton 32, 91-94 (1992). PETERSON, G. L.: A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83,346-356 (1977). PLOEGMAN, J. H., G. DRENT, K. H. KALK, W. G. J. HOL, R. L. HEINRIKSON, P. KEIM, L. WENG, and J. RUSSELL: The covalent and teniary structure of bovine liver rhodanese. Nature 273, 124-129 (1978). SCHMIDT, A.: Regulation of sulfur metabolism in plants. Progress in Botany 48, 133-150 (1986). SCHMIDT, A, I. EROLE, and B. GAMON: Isolation and characterization of thiosulfate reductases from the green alga Chlorella JUsca. Planta 162, 243-249 (1984). SCHMIDT, A, I. EROLE, and H.-P' K6ST: Changes of C-phycyanin in Synechococcus 6301 in relation to growth on various sulfur compounds. Z. Naturforsch. Tell C 37, 870-876 (1982). S6RBo, B. H.: Rhodanese. Meth. Enzymol. 2, 334-337 (1955). SYKES, A. H.: Early studies on the toxicology of cyanide. In: VENNESLAND, CONN, KNOWLES, WESTLEY, and WINING (cds.): Cyanide in Biology. Academic Press, London (1981). THOMAS, D., R. BARDEY, D. HENRY, and Y. SUROIN-KERJAN: Physiological analysis of mutants of Saccharomyces Ctrevisiat impaired in sulphate assimilation. J. Gen. Microbiol. 138, 2021-2028 (1992). TRUPER, H. G. and N. PFENNIG: Sulphur metabolism in Thiorhodaceat. III. Storage and turnover of thiosulphate in Thiocapsa floridana and Chromatium species. Antonie van Leewenhoek J. Microbiol. Serol. 32,261-276 (1966). UHTEG, L. C. and J. WESTLEY: Purification and steady-state kinetic analysis of yeast thiosulfate reductase. Arch. Biochem. Biophys. 195,211-222 (1979). WESTLEY, J.: Cyanide and sulfane sulfur. In: VENNESLAND, CONN, KNOWLES, WESTLEY, and WINING (cds.): Cyanide in Biology. Academic Press, London (1981).