AbstractMembranes of the green sulfur bacterium, Chlorobium limicola f, thiosulfatophilum, catalyze the reduction of externally added isoprenoid quinones by sulfide. This activity is highly sensitive to stigmatellin and aurachins. It is also inhibited by 2-n-nonyl-4-hydroxyquinoline-N-oxide, antimycin, myxothiazol and cyanide. It is concluded that in sulfide oxidizing bacteria like Chlorobium, sulfide oxidation involves a sulfide-quinone reductase (SQR) similar to the one found in Oscilatoria limnetica [Arieli, B., Padan, E. and Shahak, Y. (1991) J. Biol. Chem. 266. 104–111]

Arieli, B.

Hauska, G.

Padan, E.

Shahak, Y.

Elsevier - Publisher Connector 

Volume 299, number 2, 127-130 FEBS 10795 
0 1992 Fe&ration of Eutopcan Biochemical Sociclics 00145793/92/S5.00 
March 1992 
Sulfide quinone reductase (SQR) activity in Chlorobium 
Y. Shahak”, B. Arielib**, E. l?adanb and G. Hauska’ 
“Bioclterttisrry Deparrmenr, Titc Wektrtartn lnsrirure of Sciertce, Reltnvot 76fOO. Israel, “Division of Microhhl artd Mokculur Ecology, 
Imrirure of Ltf.‘. Sciences, The Hebrew University of Jer!rsdcnt, J~rusoictrt 91904. Israel and cItts~i~u~ fiir Zelibiologie urtd 
PJurt~en~lt~Jsio~o~ie, Lhtivc~si~iit Regettsburg. 8400 Regeasburg, Germ-my 
Received 20 January 1992 
Membranes of the green sulfur bacterium. Clrlorobiu,~ lhticoh f. rltiostr~uto~lriit~r,t. catalyze the reduction of externally added isoprcnoid quinones 
by sultide. This activity is highly sensitive to stigmatellin and aurachins. It is also inhibited by 2-n-nonyl4hydroxyquinolinc&oxidc, anlimycin. 
myxothiazol and cyanide. II is concluded that in sulfide oxidizing bacteria like Ct’tlorobittttr, sulfide oxidation involves a sulfidequinone reductasc 
(SQR) similar to the one round in Oscilurori~t littmcticu [Atieli. B.. Padan. E. and Shahak, Y. (1991) J. Biol. Chrm. 266, 104-11 I]. 
Sulfide oxidation; Quinonc reduction: Electron transport; Photosynthesis; C/dorobiut)r hticoh 
1. INTRODUCTION 
The Chlorobicrceae are obligate photoautotrophic 
green bacteria, able to use H,S and So. Chlorobittrtt 
hicola f. riziostrlfrrropltilurlz can also utilize thiosulfate 
(S,Oi-). The intermediates observed uring H2S oxida- 
tion by Chlorobiunz are thiosulfate and So, the final 
oxidation product being sulfate (SO:-) (see [I] for a 
review). 
The initial step in sulfide electron donation into the 
photosynthetic electron transport chain is a matter of 
debate. In several species of phototrophic bacteria, the 
sulfide-oxidizing enzyme was proposed to be flavocy- 
tochrome c. Indeed, flavocytochrome c,which has been 
found and isolated from several sulfur bacteria ([2]. see 
also [l]), catalyzes electron transfer from I&S to soluble 
cytochrome c. However the role of flavocytochrome c 
as the major initial step in sulfide oxidation has recently 
been questioned, because it is absent from several 
sulfide-oxidizing bacteria ([3], see also [l]). Alterna- 
tively, the transfer of electrons from sulfide primarily 
into the quinone pool was proposed [1,4]. 
twica, which is the most thoroughly studied species, can 
readily shift from oxygenic (plant-type) to anoxygenic 
photosynthesis. The shift is induced by sulfide under 
light and anaerobic onditions, and depends on the syn- 
thesis of new proteins [5,6]. Sulfide-dependent CO: fixa- 
tion involves the cytochrome bbf complex and photo- 
system 1, but not photosystem II [6-81. Light-dependent 
sulfide oxidaticn is maintained in isolated, washed thy- 
lakoids prepared from induced cells [93. Recently we 
have found that the induced thylakoids can specifically 
catalyze sulfide-dependent reduction of externally 
added quinones, in the dark. We proposed that a novel 
electron carrier, sulfide-quinone reductase (SQR), is the 
inducible protein catalyzing the initial step in sulfide 
oxidation in 0. limrteticu [lo]. The SQR was further 
solubilized from induced 0. fimrteticcr thylakoids. and 
purified. The isolated enzyme maintains its high affinity 
to sulfide and quinone, as well as it< differential sensi- 
tivity to inhibitors (Arieli, Shahak and Padan, in 
preparation). 
Sulfide-dependent anoxygenic photosynthesis occurs 
also in several species of cyanobacteria. Oscillaroria Ikt- 
In the present work we report on the occurrence of 
SQR also in Chlorobium and suggest he universality of 
SQR as the enzyme catalyzing sulfide-quinone oxido- 
reduction in photoautotrophs. 
Corrc,sporr&ncc nddrcss: Y. Shahak, Institute of Horticulture. The 
Volcani Center, PO Box 6, Bet-Dagan 50-250. Israel. 2. MATERIALS AND METHODS 
*Ptm:ttt address: Department of Biological Sciences, Purdue Uni- 
versity, West Lafayette, IN 47907, USA. 
Abbrevirr~ions: SQR, suifide-quinone reductase; MQ, mcnaquinone; 
PQ-1(-Z), plastoquinonc-l(G); UQ-l(G), ubiquinone-I(-?); NQNO. 
3-n-non)!4hydroxyquinoline-N-oxide; UHDBT, 5+undecylb- 
hydroxy-4,7-dioxobsnzothiazole: DNP-1NT. 2-iodo-6.isopropyl-3- 
methyl-2’-4,4’-trinitrodiphcnyl ether; JXMU. 3-(3’,4’-dichlo- 
rophenyl)-l,l-dimelhylurea; Bchl c, bactcriochtorophyll c. 
Cells from Chlorohiurrt lirrricokt F. iltiosr~rophilurrt WCK! grown and 
membranes were prepared as described before [I I]. Membranes were 
stored at -20°C. Bactcriochlorophyll c conccntrdtion was determined 
from the spectra in aqueous olution, using an extinction cocfficicnt 
of 90 for I mg B&l c/ml al 750 nm. which was estimated from a 
parallel measurement in methanol [l?]. 
Spectra were measured and processed by a Kontron Uvikon 860 
spectrophotometcr equipped with a stirred and thcrmostated cuvcttc 
liolder. which was placed close to the dctcctor to minimize light scan- 
Published b)j Ehevicr Scimce Publiskers 6. V. 127 
Volume 299, number 2 FEBS LETTERS March 1992 
B 
0.5 ml87 
boiled mombranea 
i0 275 300 326 3! 
C 
(a) no membranes 
lmh 
t 
Wavelength, nm 
Fig. 1. Sulfide-dependect plastoquinone-1 reduction catalyzed by Chlorobirrrr~ membranes, Reaction mixture coniaincd 211 mM TRIS-HCI, pH 5,8, 
membranes containing 8.4 pg Bchl c/ml and either 40 yM PQ-I and 200 PM Na,S (A and B) or 2OpM PQ-I and 40 yM Na$ (C). (A) and (B) 
illustrate spectra scanned at the indicated times after PQ-i injection, after the digital subtraaicrn of the spectrum taken shortly prior to the injection. 
Each scan took 15 s for completioe. The indicated times are the starling times. In (B) the membranes were incubated for 10 min at 100°C before 
addition. (C) illustrates rcntinuous monitoring of PQ-I trduction by dual wavelength spectroscopy. Where indicated, 4PM NQNO was added 
to the membranes. For olher details see Materials and Methods, The numbers in parentheses in Fig. 1C indicate the initial quinone reduction in 
pmol/mg Bchl L’* h. 
tering effects, Dual-waselenglh absorption changes were monitored 
continuously by an Aminco DW-?a spectrophotometcr as previously 
described [IO]. 
The reactions were carricti out under N2 atmosphere in stoppered 
quartz cuvettes at 24°C. Typically. the reaction mixture (3 ml) was 
composed of 20 mM TRIS-HCI, pH 7.8,40pM quinone, 40pM Na,S, 
and membranes containing 8-10 c(g Bchl c/ml. 
Preparation of stock solutions and concentration determination of 
quinonesand quinone-analog inhibitors was done as described in [lo]. 
PQ-I was synthesized according to Rich [ 131. PQ-2, UQ- I and UQ-2 
were gifts of Dr. G. Hind, Brookhaven National Laboratory, USA, 
of Hoffmann-La Roche, Switzerland and of Dr. E. Keynan, The 
Tcchnion, Israel, respectively. Duroquinone was purchased rrom 
Aldrich, antimycin A, vitamin K, and DCMU from Sigma. The fol- 
lowing quinone-analog inhibitors were generously donated: stigmatel- 
lin, and aurachin C and D by Dr. G.H. Halle, Braunschweig, Gcr- 
many; NQNO and UHDBT by Dr. B.L. Trumpower, Dartmouth 
Medical School, Hanover, USA; and DNP-INT by Dr. A. Trebst, 
Ruhr University, Bochum, Germany. 
3. RESULTS AND DISCUSSION 
Chhobium membranes catalyze the electron transfer 
from sulfide to externally added quinones, in the dark. 
Fig. 1 illustrates ulfide-dependent PQ-1 reduc:ion. The 
spectral changes indicate that PQ-1 became fully re- 
duced within less than 10 min of incubation with sulfide 
and Cithobiunt membranes, under the experimental 
conditions (Fig. 1A). Fig. 13 shows that with boiled 
membranes, the spectral changes developed much 
slower. The rate of the enzymatic reaction, unlike the 
boiled control, decreased with time (compare Fig. 1C. 
traces (a) anti (c)). XQNO, a quinone anaiog which has 
previously been found to specifically inhibit SQR activ- 
ity in 0. hnerica [lo], also inhibited it in Chlorobium 
(Fig. lC, trace (b)), further supporting the enzymatic 
nature of the reaction. NQNO had no effect on the rate 
with boiled membranes (not shown). 
In addition to the enzymatic reaction, Chlorobium 
membranes eem to contain an unknown, heat-stable 
substance which can stimulate sulfide-quinone oxido- 
reduction. The initial rates of qainone reduction by 
sulfide, measured in the presence of boiled membranes, 
was markedly faster than that in the absence of 
membranes. For example, thr ratio of the rate with 
boiled membranes to the one in absence of membranes 
in Fig. 1C is about 4 (traces (a) and (c)). This ratio 
further increased upon increasing the substrate cnn- 
centrations. In accordance, also the NQNO-insensitive 
portion of quinone reduction by sulfide in intact 
membranes was much more pronounced at high sub- 
strate concentrations. These background rates did not 
decrease with time, but the enzymic reaction did (Fig. 
lC, trace (a)). Furthermore, at higher sulfide concentra- 
tions (>200yM) the reduction of quinone in the absence 
of membranes increased subsrantially within the 
measuring time of a few minutes. Probably, chemical 
reduction of quinones is autocatalytically stimulated by 
the accumulation of small amounts of semiquinone dur- 
ing the course of the reaction [14]. Therefore, we 
avoided using sulfide and quinone concentrations 
higher than 40 PM, and we corrected SQR rates for the 
rates obtained with boiled membranes. It is worth 
mentioning that in 0. iitnnefica the initial rates of 
sulfide-dependent PQ-1 (or PQ-2) reduction measured 
in the absence of membranes or with boiled membranes 
are comparable [lo]. 
In the experiments ummarized in Table I we have 
tested several externally added quinones for their activ- 
ity with Chlorobiurn SQR. Ubiquinone and plastoqui- 
1% 
Volume 299, number 2 FEBS LETTERS March 1992 
Teblc I 
Chlorobiwrt SQR activity with different quinone acceptors 
Quinone acceptor SQR initial rate 
Wmol Q red/m6 Bchl c’h) 
Vitamin K, 0 
Mcnadionc 1.9 
2mmethyl-3-mcthylthio. I .4- 
naphto-quinonc 0.8 
UQ-1 5.0 
UQ-2 15.9 
PQ- I 17.5 
PQ.2 27.4 
Duroquinonc I.3 
The reaction mixture was as described for Fig. I, with quinones prcs- 
ent at 20 PM. Sulfide concentration was 56 pm. The following coeffi- 
cients were used (for I mM): 14.8 for vitamin K,, menadionc and 
mcthylthio mcnadione (at 295-270 nm): 16.9 for PQ- I and PQ-2 (at 
292-26G nm); 17.8 for UQ-I or UQ-2 (at 300-280 nm); and 18.0 for 
duroquinonc (at 295-272 nm; see [IO]). Rates were corrected for con- 
trols with boiled membranes, 
none, which have short isoprenoid side chains, were 
good external acceptors. In both cases a side chain com- 
posed of two isoprenoid units was preferable to one 
unit. Duroquinone, which lacks an extended side chain, 
was a relatively poor acceptor. Since the endogenous 
quinone pool in Cidorobium consists of MQ-7 and 1’. 
oxo-MQ-7 (Cltlorobiutn quinone; see [15]), we also 
tested 3 naphtoquinones. However, they gave no, or 
only poor, reduction rates (Table 1). Menadione and 
2-methyl-3-methylthio-1,4-naphtoquinone, like duro- 
quinone, lack an extended side chain, and vitamin KI 
(phylloquinone) may be too insoluble to partition fast 
enough into the membranes. Alternatively, if C%lorc+ 
bium quinone, with its high redox potential [15] func- 
tions as the physiological electron acceptor, the redox 
potential of 2,3-substituted I ,4-naphtoquinones may be 
too low [15,16], and the ones of PQ and UQ may just 
suffice [17,18]. It is worth mentioning in this context 
Table II 
Inhibitors of SQR activity in ClllovuBi~~ 
Inhibitor 
- I50 (nM) 
Stigmatellin 5 
Aurachin C I2 
Aurachin D 38 
NQNO 160 
Antimycin A 960 
Myxothiazol 6,000 
KCN I0,000 
UHDBT No ciTect 
DNP-INT No effect 
DCMU No effect 
Reaction conditions were as for Fig. IC, IsI, is the inhibitor concentra- 
tion required for 50% inhibition of SQR initiul rate. The inhibitors 
were added to the reaction mlxttlre containing the membranes (while 
stirring), l-2 min prior to the sequential addition of PQ-I and Na,S. 
that electron flow from succinate to MQ is energy-de- 
pendent in bacilli [19], and therefore is attenuated in 
non-vesicu!ar, cell-free systems. 
The Chlorobim SQR activity was extremely sensitive 
to three naturally occurring inhibitors (Table II): stig- 
matellin. and aurachin C and D. They inhibit electron 
transfer at th: QB site of photosystem II and at the 
cytochrome bdcomplex, as well as at the mitochondrial 
and bacterial cytochrome bc, complex [20-221. Under 
our experimental conditions, the Cklorobim SQR ac- 
tivity was sensitive to nanomolar concentrations of the 
inhibitors, with stigmatellin the most potent (Iso = 5 nM; 
pIso = 8.3), No other system studied so far is as sensitive 
to stigmatellin. NQNO and the two other antibiotics, 
antimycin and myxothiazol, inhibited at ,uM concentra- 
tions. 
It is of interest o compare the inhibitor sensitivity of 
Chlorobiutn SQR with the 0. limwticu SQR. The au- 
rachins inhibit the cyanobacterial enzyme qually well 
(Arieli, Shahak and Padan, in preparation), but stigma- 
tellin is by far a superior inhibitor of the Chlorobim 
enzyme. Unlike in Cldorobim, in 0. lirnnerica the SQR 
is not inhibited at all by either antimycin or myxothiazol 
[lo]. Also KCN only inhibits the Chlorobium SQR. The 
opposite is true for UHDBT, which is a fairly good 
inhibitor in 0. Iktrtetica. SQR in both systems is insensi- 
tive to DNP-INT or DCMU. The differences between 
the two systems might reflect differences in the quinone 
binding sit.e of each enzyme, which might be related to 
the different endogenous quinone acceptor in each sys- 
tem. 
The observed rates of SQR in CMorobium mem- 
branes and the rates reported for the reduction of cy- 
tochrome c by sulfide via flavocytochrome c-553 of 
Chlorobim are comparable, corresponding to some 10 
s-l in TON. Since the Ravocytochrome is largely washed 
out from our meriibl;anes we conclude that sulfide oxi- 
dation in Chlorobium is not exclusively occurring via 
flavocytochrome c, but also via SQR and the quinone 
pool [1,4], as in Oscifiaroricc. This path may be more 
tightly linked to energy conservation. 
,ick,~c~~~irdgref~?aru: Dr. A. Trebst, Bochum, and Dr. H.G. Trtlper, 
Bonn, are highly acknowledged for helpful discussions, the latter also 
for handling the transportation of frozen Cll!orobiur~t membranes 
from Germany to Israel. This work was supported by the Cicrman- 
Israel Foundation for Scientific Research and Dcvclopmcnt (GIF). 
Grant 1-91-l 18.9188. 
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Sulfide quinone reductase (SQR) activity in Chlorobium 

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Sulfide quinone reductase (SQR) activity in Chlorobium

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