Mechanism of nitrite reduction to nitrous oxide in a photodenitrifier, Rhodopseudomonas sphaeroide forma sp. denitrificans

Biochimica et Biophysica Acta 841 (1985) 201-207

201

Elsevier BBA 22102

Mechanism of nitrite reduction to nitrous oxide in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitriflcans

Katsuro Urata and Toshio Satoh Department of Biology, Faculty of Science, Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya-ku, Tokyo 158 (Japan) (Received March 18th, 1985)

Key words: Cytochrome bc1 complex; Nitric oxide reductase; Nitrite reductase; Nitrous oxide; Photodenitrifier

The mechanism of anaerobic reduction of NO 2 to N20 in a photodenitrifier, Rhodopseudomonas splmeroides forma sp. dem'trificans, was investigated. With ascorbate-reduced phenazine methosulfate (PMS) as the electron donor, the nitrite reductase of this photodenitrifier reduced NO2- to NO and a trace amount of N20. With dithionite-reduced benzyl viologen as the electron donor, the major product of NO 2- reduction was NHzOH, and a trace amount of N20 was also produced. The nitrite reductase itself had no NO reductase activity with ascorbate-reduced PMS. It was concluded that the essential product of NO 2 reduction by the purified nitrite reductase is NO. Chromatophore membranes stoichiometrically produced N20 from NO2-- with any electron donor, such as dithionite-reduced benzyl viologen, ascorbate-reduced PMS or N A D H / F M N . The membranes also contained activity of NO reduction to N20 with either ascorbate-reduced PMS or duroquinol. The NO reductase activity with duroquinol was inhibited by antimycin A. Stoichiometric production of N20 from NO2- also was observed in the reconstituted NO2reduction system which contained the cytochrome bc ! complex, cytochrome cz, the nitrite reductase and duroquinol as the electron donor. The preparation of the cytochrome b c l complex itself contained NO reductase activity. From these results the mechanism of NO2- reduction to N20 in this photodenitrifier was determined as the nitrite reductase reducing NO2-- to NO with electrons from the cytochrome bc ~ complex, and NO subsequently being reduced, without release, to N20 with electrons from the cytochrome bc t complex by the NO reductase, which is closely associated with the complex.

Introduction Denitrification is the sequential reduction of N O f to volatile products (N20 or N 2). The process is described as follows: NO 3 -" NO 2 "* (NO'?.) -~ N20 --~ N 2

In the course of denitrification, the details of the process by which two NO 2 anions are reduced to produce N20 (a four-electron reduction) are still Abbreviations: PMS, phenazine methosulfate; DDC, N,N-diethyldithiocarbamate.

the subjec t of controversy [1-5]. The discussion has been focused on whether NO 2 is reduced directly to N 2 0 , o r whether NO is formed as a free intermediate that is subsequently reduced to N20. There have been many reports favouring the latter. Low levels of NO were observed directly during NO 2 reduction [1,6,7]. The exchange of nitrogen between isotopically labelled NO2 and a pool of added NO was observed during reduction of NO 2 to N 2 0 [1]. EPR measurements showed that a heme-NO complex was formed in purified hemecontaining nitrite reductase [8]. The product of N O f reduction by the purified nitrite reductase

0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

202 was reported to be N() with ascorbate-redu~,cd PMS or tetramethyl-p-phenylenediam ne [9 I 1 i contrast, recent reports showed that the pn,,:< ,'; of N O r reduction by the purified reduct;,: . . . . ! Ak'aligenes sp. are identified as N H +()H. P'; ~i t~vi N H 4 with dithionite-reduced methyl vi,+!, ,. and that free N O in cultures of P,,, , ~ , nitrificans and Pseudomonas aerm!:.~ N ( L reduction could r|ot be detec{c+Li i The problems mentioned abo~c ~t~v ..+,>nce:+,:u with whether NO+ reduction to Net is catalyzed only by nitrite reductase, or whether N() reductase participates. It has been reported that roans purified nitrite reductases also reduce N O In N+() 19,11,13], and Averill and Tiedje [141 proposed that the cytochrome cd-type nitrite reductase can reduce NO2 immediately to NA). In contrast. Payne [2,3] postulated that N O is a free intermediate and that the N O reductase distinct from the nitrite reductase participates in N()+ reduction to N,()+ However, the entity of tile N() reductase remains undefined. Sawada et al. [101 purified and characterized a nitrite reductase of a photodenitrifier, Rhodopseudomonas sphaeroide,r forma sp. denitr(/ican.s" [15]. They identified the product of N O , reduction as N O with ascorbate-reduced PMS as the electron donor. We have shown [16] that the N()~ reduction system is reconstituted with the cvtochrome bc~ complex, cytochrome ,'~ and nitrite reductase and that reducing equivalents are transferred from the c y t o c h r o m e h('~ complex to nitrite reductase by way of cytochrome (',. In the present study, we have used the in vitro reconstituted NO+ reducing system and studied the mechanism ~t" N O , reduction to N~()+ T

,)

Materials and Methods

Growth conditions and preparation of chromatophore membranes. R. sphaeroides forma sp. denitrificans IL-106 [15] was grown under denitrifying conditions at 30°C in light as described previously [10]. Cells were harvested at an early stationary phase of growth, washed twice with 50 mM potassium phosphate (pH 7.0) and then suspended in a 3-fold volume of the same buffer. The cell suspension was sonicated at 20 kHz for 2 rain, and chromatophore membranes were obtained by

subjecting the sonicatc to differentiui ccntrlttlga+ :: ~,~for 20 rain +lllt+] 1()5 t)+[){)>, for 60 n, in+ "['he nmmbranes v, erc suspended in s rnM potasshun phosphate (pH 7.0) to ~,btain a concentration of 3.62 rng protein/: ml. I+:+dation of the cl'lochrome he] c,mp/c+v, cvlo,/u'o,;c c, and t/re nitrite rcdz,'la.s'e. The tyrochrome h++t complex, c\'tochrome ,.~ and the nitrite +eductasc v, ere isolated as described previously -

,

,

o +~

+lssal'.+. All the enzyme activities were +.teD ( urtder argon. NO, reduction 1) with a gas-tight syringe at 3-min intervals, and N+,O was determined by gas c h r o m a t o g r a p h y equipped with an electron capture detector [18]. N O reduction acti`+:itv was assayed by determining the amount of N+O produced in the same reaction medium for NO; reduction, except that 0.1 mM D D C "+,+'asadded to inhibit NO+ reductiorl [10], since N O reacts with contaminated oxyger,, to produce nitrous acid (NO2) which hydrates irreversibly at pH 7.0 to form NO{ and NO, 11], In this case. only ascorbate-reduced PMS or duroquinol was used as the electron donor. since a non-enzymatical formation of N~O from N O ;va>, observed with dithionite-reduced benzvl viologen tw N A D H / ' F M N (data not shown). The reaction `+',.as initiated bv adding N() gas to 3~4 (v/:v) of the gas phasc. At 3-rain intervals, gas sanrples (50 ptl) were taken and then N~O was determined by gas c h r o m a t o g r a p h y [18]. N O was determined by gas c h r o m a t o g r a p h y equipped with a thermal conductivity detector. Aliquots (50 btl) of the gas phase were injected onto a 5-m column of Porapak Q at 40°C and eluted with argon. When N H 2 O H was determined, N H 2 O H was oxidized with an acidic iodine solution to NO-, [19] which was determined by the method of Nicholas and Nason [17 I. o

•

203

Other determination. Protein content was determined by the method of Lowry et al. [20] modified by Hartree [21]. Cytochrome content was determined from the reduced-minus-oxidized difference spectrum with EmM values of 20.0 and 19.1 at the peaks of the a-band for b- and c-type cytochromes, respectively [22]. Materials. Duroquinol was prepared from duroquinone (Tokyo Chemical Industry Co.) described by Rich [23] and stocked in 25 mM in 96% ethanol with 10 mM HC1. Antimycin A, benzyl viologen and NADH were purchased from Sigma Chemical Co. Phenazine methosulfate, FMN, and NH2OH. HC1 were obtained from Wako Pure Chemical Industries. Commercial NO (Kurosawa Asanki Co.) was purified as described before [24]. NO was sparged through 1 M K2HPO 4 and collected over deoxygenated 1 M K2HPO4 in a closed vial in order to remove contaminating nitrous acid (NO2) which hydrates irreversibly at pH 7.0 to form NO 2 and N O / . Purified NO was transferred to reaction cuvettes with a gas-tight syringe which had been purged with argon. Results

Product of N O r reduction by isolated nitrite reductase with artificial electron donors The discussion concerning the mechanism of NOE reduction to N20 has been focused on whether NO 2 is reduced directly to NEO by nitrite reductase, or whether NO is formed as a free intermediate by the nitrite reductase and subsequently reduced to N20 by NO reductase. First, the products of NOE reduction by the purified nitrite reductase of this photodenitrifier were examined with artificial electron donors (Table I). With ascorbate-reduced PMS, NOr was almost stoichiometrically reduced to NO, and only a trace amount of N20 was detected; this is quite consistent with our previous report [10]. When dithionite-reduced benzyl viologen was used, most of the product was NHzOH and NO was not detectable by gas chromatography. The NHzOH was presumed by the following experiment to be produced by a non-enzymatic reaction: when 21 #mol of NO (1% of the gas phase) was introduced into a 50 ml serum bottle with a serum cap containing 5 ml of argon-saturated 50 mM potassium phos-

TABLE I PRODUCTS OF NO{ REDUCTION BY THE PURIFIED NITRITE REDUCTASE WITH ARTIFICIAL ELECTRON DONORS 1 ml of the reaction medium was introduced in a 12 ml serum bottle equipped with a serum rubber cap. When ascorbate-reduced PMS was used as the electron donor, the reaction medium consisted of 50 mM potassium phosphate (pH 7.0), 50 mM KNO 2, 10 mM sodium ascorbate, 0.1 mM PMS and 5/~M nitrite reductase. The products were determined when NO 2 was exhausted after 30 rain incubation at 25°C under argon. When dithionite-reduced benzyl viologen was used, the reaction medium consisted of 50 mM potassium phosphate (pH 7.0), 0.2 mM KNO 2, 50 mM dithionite, 1 mM benzyl viologen and 1.7 /LM the nitrite reductase. The products were determined when NO~- was exhausted after 3 min incubation at 25°C under argon, n.d., not detectable. Electron donor

Ascorbate-reduced PMS Dithionite-reduced benzyl viologen

Yield from N O / consumed (%) NO

NH2OH

99.5

n.d.

N20 0.45

n.d.

89.7

< 0.01

phate (pH 7.0)/50 mM dithionite/1 mM benzyl viologen, about 2.6 #mol of NH2OH and a trace amount of N20 were produced after 25 min incubation at 20°C. The trace amount of N 2 0 detected with either electron donor was presumably derived from a non-enzymatic reaction of NHEOH with NO, or from a nitrite reductase-mediated reaction of NH2OH with NO 2 [25]. It was concluded from these results that the product of the nitrite reductase is NO.

The product of N O r reduction by chromatophore membranes with artificial electron donors The product of the NO 2 reduction by intact cells with endogenous substrates is N20 when N/O reduction is blocked by acetylene or azide [26,27], and neither NO nor NH20H is detectable. Since the discrepancy of products of NO 2- reduction between intact cells and the purified nitrite reductase could conceivably be attributed to the artificial electron donors used and to the presence or absence of membrane fractions, the amounts of N 2 0 produced and NO 2 consumed by chromatophore membranes with artificial electron donors were determined (Table II). Even when dithionitereduced benzyl viologen or ascorbate-reduced PMS

204 TABLE II

TABLE IlI

STOICHIOMETRIES OF NO 2 CONSUMPTION AND N20 PRODUCTION IN NO~- REDUCTION BY CHROMATOPHORE MEMBRANES WITH VARIOUS ELECTRON DONOR SYSTEMS

NO2 CONSUMPTION AND N20 PRODUCTION IN THE RECONSTITUTED NO 2 REDUCTION SYSTEM

The reaction medium consisted of 50 mM potassium phosphate (pH 7.0), 0.5 mM KNO 2, chromatophore membranes (0.72 mg/ml), and a given electron donor system. The electron donors were 50 mM dithionite/1 mM benzyl viologen, 10 mM sodium ascorbate/0.1 mM PMS, and 1 mM NADH/5 mM FMN. NO 2 consumption and N20 production were determined and expressed as nmol/mg protein per min. Electron donor system

NOy consumed

N20 produced

N20 / NO 2

Ascorbate-reduced PMS Dithionite-reduced benzyl viologen NADH + FMN

32.2

18.1

0.56

34.4 23.6

17.5 11.1

0.51 0.47

bc 1

complex/nitrites reductase (nmol cyt. c1/nmol)

was used, the N 2 0 / N O 2- ratio was a l m o s t 0.5, i n d i c a t i n g that N 2 0 was p r o d u c e d a c c o r d i n g to the following e q u a t i o n : NO 2 + 3 H + + 2 e - ~ 0 . 5 N 2 0 + l . 5 H 2 0

The reaction medium consisted of 50 mM potassium phosphate (pH 7.0), 0.05% sodium cholate, 0.15 /LM cytochrome bc1 complex, 38 /,tM cytochrome c2, 100 /xM duroquinol and various amounts of nitrite reductase. NO~ and N20 were determined after 20 min incubation at 25°C under argon. When NO 2- was determined, the reaction was terminated by adding 20/d of 100 mM ferricyanide.

(1)

This is quite consistent with the result that the H + c o n s u m p t i o n stoichiometry, H + / N O 2 - ratio, of a cell suspension of this p h o t o d e n i t r i f i e r is - 3.18 in the N O 2 r e d u c t i o n to N 2 0 with dithionite-red u c e d benzyl viologen as the electron d o n o r [28]. W e have f o u n d that N A D H / F M N is a g o o d electron d o n o r system for N O £ r e d u c t i o n of chromatophore membranes. When NADH/FMN was used, stoichiometric p r o d u c t i o n of N 2 0 was also observed. These results suggest that the p r o d u c t of N O 2 r e d u c t i o n in c h r o m a t o p h o r e m e m b r a n e s is N 2 0 , i n d e p e n d e n t of the electron d o n o r s used, a n d that the m e m b r a n e s are required for the stoichiom e t r i c p r o d u c t i o n of N 2 0 .

Participation of the cytochrome bc I complex in N20 production from N O 2 W e have r e p o r t e d that the N O 2 r e d u c t i o n system is r e c o n s t i t u t e d with the c y t o c h r o m e bc t complex, nitrite reductase, c y t o c h r o m e c 2 a n d d u r o quinol as the electron d o n o r [16]. Then, N 2 0 p r o d u c t i o n f r o m N O 2 in the r e c o n s t i t u t e d system was examined. N 2 0 was p r o d u c e d linearly with time in the c o m p l e t e r e c o n s t i t u t e d system a n d n o N 2 0 was d e t e c t e d in the system w i t h o u t the cyto-

2.0 1.0 0.1 0.05 0

NO2 consumed (nmol)

N20 produced (nmol)

N20// NO 2

55.5 114.2 64.7 74.7 41.8

27.8 57.2 14.1 14.8 0

0.50 0.50 0.23 0.20 0

c h r o m e bc 1 c o m p l e x ( d a t a not shown). Since the results suggested the possibility that the cytoc h r o m e bc 1 c o m p l e x plays an i m p o r t a n t role in the N 2 0 p r o d u c t i o n from NO2-, stoichiometries of N O 2 c o n s u m e d a n d N 2 0 p r o d u c e d in the reconstituted system with various ratios of c y t o c h r o m e bCl c o m p l e x to nitrite reductase were e x a m i n e d ( T a b l e III). The c o n c e n t r a t i o n of c y t o c h r o m e c 2 was much higher t h a n those of the nitrite red u c t a s e a n d the c y t o c h r o m e bc I complex. W h e n the ratio of the nitrite reductase a n d the cytoc h r o m e bc t c o m p l e x was a b o v e 1, N O 2 was red u c e d to N 2 0 stoichiometrically a c c o r d i n g to Eqn. 1. O n the other hand, when the ratio was b e l o w 1, the ratios of N 2 0 / N O 2 were less t h a n 0.5, a n d no N 2 0 was p r o d u c e d in the a b s e n c e of the complex, i n d i c a t i n g the f o r m a t i o n of a n o t h e r p r o d u c t ( p r o b a b l y N O ) t h a n N 2 0 by direct electron flow from d u r o q u i n o l to c y t o c h r o m e c 2. These results suggest that the c y t o c h r o m e bc t c o m p l e x is i n d i s p e n s a b l e in the f o r m a t i o n of N 2 0 .

Existence of N O reductase activity in the cytochrome bc I complex Averill a n d T i e d j e [14] p r o p o s e d the h y p o t h e s i s that the c y t o c h r o m e cd-type nitrite reductase has N O r e d u c t i o n activity a n d can reduce N O 2 to N 2 0 for itself. In this p h o t o d e n i t r i f i e r , however,

205 TABLE IV ACTIVITIES OF NO~- REDUCTION TO N20 AND NO REDUCTION TO N20 IN THE RECONSTITUTED SYSTEM The complete reaction medium consisted of 50 mM potassium phosphate (pH 7.0), 0.05% sodium cholate, 32 #M cytochrome c2, the cytochrome bcI complex (0.15 #M cytochrome cl), 2.3 #M nitrite reductase, 10 mM sodium ascorbate and 0.1 mM PMS. The reaction was initiated by adding 0.13 mM KNO2 or NO gas to 3% (v/v) of the gas phase. The activities were assayed and expressed as nmol NO 2 produced/min. Components of reaction medium

NO 2 reduction to N20

NO reduction to N20

- DDC

+ DDC

- DDC

+ DDC

3.6 0 0

0 0 0

3.7 3.7 0

3.1 3.3 0

bc1 complex+cyt.cI + nitrite reductase

bcI complex cyt.c2 + nitrite reductase

the p u r i f i e d nitrite reductase p r o d u c e d N O f r o m NO~-, a n d N 2 0 p r o d u c t i o n was o b s e r v e d o n l y in the presence of c y t o c h r o m e b q complex. T a b l e IV shows activities o f NO~- r e d u c t i o n to N 2 0 a n d N O r e d u c t i o n to N 2 0 in a m i x t u r e of various c o m p o n e n t s with D D C as an i n h i b i t o r against the nitrite reductase a n d a s c o r b a t e - r e d u c e d P M S as the electron d o n o r . T h e activity of NO~- r e d u c t i o n to N 2 0 was o b s e r v e d only in the c o m p l e t e m i x t u r e c o n t a i n i n g the c y t o c h r o m e bc~ complex, cytoc h r o m e c2 a n d the nitrite reductase, a n d was in-

TABLE V SUBCELLULAR DISTRIBUTION OF NO AND NITRITE REDUCTASE ACTIVITIES WITH ASCORBATE-REDUCED PMS AS AN ELECTRON DONOR Cells from 2300 ml culture under denitrifying conditions in light were used for the preparation of chromatophore membranes and soluble fraction. The reaction medium contained 50 mM potassium phosphate (pH 7.0), 10 mM sodium ascorbate, 0.1 mM PMS and a fraction (from 0.5 to 0.6 mg protein/ml). The reaction was initiated by adding NO gas to 3% (v/v) of the gas phase or 0.5 mM KNO 2. Fraction

NO reductase activity

Nitrite reductase activity

specific a

total b

specific c

total a

Chromatophore membranes

27.5

3025

17.3

1133

Soluble fraction

1.5

284

37.7

8803

a b c d

nmol N20 produced/min per mg protein. nmol N20 produced/min. nmol NO 2 consumed/min per rag protein. nmol NO 2 consumed/rain.

h i b i t e d b y D D C . O n the other hand, N O reductase activity was d e t e c t e d b o t h in the c o m p l e t e system a n d the c y t o c h r o m e bc 1 c o m p l e x alone, a n d it was n o t sensitive to D D C . T h e different sensitivity to D D C of the two activities indicates that N O red u c t a s e is distinct from the nitrite reductase. This also was s u p p o r t e d b y there being no p r o d u c t i o n o f N 2 0 from N O in the absence of the c y t o c h r o m e bcl complex. These results suggest that the prep a r a t i o n s of the c y t o c h r o m e bc t c o m p l e x f u n c t i o n in the r e d u c t i o n of N O to N 2 0 . T a b l e V shows the subcellular d i s t r i b u t i o n of N O reductase a n d nitrite reductase activities with a s c o r b a t e - r e d u c e d P M S as the electron donor. It is clear that the N O reductase activity actually exists exclusively on the c h r o m a t o p h o r e m e m b r a n e s a n d that N O 2- r e d u c t a s e activity exists in the soluble fraction as r e p o r t e d before [29]. These results are also consistent with the existence of the N O red u c t a s e activity in the p r e p a r a t i o n s of the cytoc h r o m e bcl complex.

Involvement of the cytochrome bcI complex in the electron transfer for N O reduction It is conceivable that the c y t o c h r o m e bc 1 c o m plex was involved in the electron transfer of N O r e d u c t i o n if N O reductase was on the complex. Then, the effect of a n t i m y c i n A on the N O reduction in c h r o m a t o p h o r e m e m b r a n e s p r e p a r e d from cells grown u n d e r p h o t o s y n t h e t i c a n d denitrifying c o n d i t i o n s was e x a m i n e d with d u r o q u i n o l as the electron donor. A n t i m y c i n A inhibits the N 2 0 p r o d u c t i o n f r o m N O , i n d i c a t i n g that the reducing equivalents were delivered to N O t h r o u g h the cy-

206

["% I

I l l

|

l

T !

I

% l l l l

Z/A : 0 . 0 0 3

%

% l l

l l l l l

NO reductase, but that inducible peptide with NO reductase activity is closely associated with the complex. Under denitrifying conditions, NO reductase activity was measured at 1.90 and 4.53 nmol N 2 0 p r o d u c e d / m i n per mg protein, the presence and absence of antimycin A, respectively; under photosynthetic conditions, the corresponding values were 0.34 and 1.68. That oxidations of both b- and c-type cytochromes by addition of 3% (v/v) NO in the gas phase were observed in chromatophore membranes (Fig. 1) also supports the role of the complex in electron transfer in the NO reduction. These findings are consistent with the report by Satoh [24] that NO itself inhibits NO reduction, as well as NO 2 reduction and photosynthetic cyclic electron transfer by blocking electron transfer in the complex, and that the catalytic site of NO reduction is distinct from the inhibitory site of NO. Discussion

l.

_..J..............."1..............1 ............. 4 550

560

Wavelength ( n m )

Fig. 1. Succinate reduced minus NO oxidized difference spectra of chromatophore membranes prepared from cells grown under denitrifying conditions. Difference spectra were measured at 25°C with a split-beam spectrophotometer (Hitachi 356). Chromatophore membranes (0.58 mg protein/ml) were incubated anaerobically with 50 mM sodium succinate and 0.1 mM DDC in 50 mM potassium phosphate (ph 7.0) in 1-cm optical pass cuvettes for 30 min, and then the difference spectra were recorded 1 min after the addition of 3% (v/v) NO in the gas phase of the test cuvene (solid line) or KNO 2 (3 mM) (dotted line), Ferricyanide (2 raM) was used to check the maximum level of cytochromes oxidized (dashed line). The absorbance was corrected by subtracting the base line absorbance.

tochrome bc 1 complex. The NO reductase appeared to be induced under denitrifying conditions, suggesting that the complex itself is not the

Concerning the in vivo pathway of NO 2 reduction to N20, it has been a question whether NO is a free obligatory intermediate or not. Hollocher and his co-workers [1,5] concluded from isotope and kinetic studies that NO is not a free obligatory intermediate in NO 2 reduction t o N 2 0 by Pseudomonas aeruginosa cells. In this case, only nitrite reductase is believed to take part in NO2- reduction to N20. One of the results supporting this hypothesis is that many purified nitrite reductases also reduce NO to N20 [9,11,13]. Recently, Averill and Tiedje [14] have proposed that the cytochrome cd-type nitrite reductase can reduce NO~ immediately t o N 2 0 and suggested that this hypothesis is also applicable to copper-containing nitrite reductases, such as the reductase of this photodenitrifier. However, this is not the case in the photodenitrifier, since the major product of NO 2 reduction by the nitrite reductase was identified as NO and the reductase itself had no activity of NO reduction. On the other hand, Payne [2,3] postulated that the reduction of NO 2 t o N 2 0 in Pseudomonas perfectomarinus involves NO as a free intermediate and that there exists NO reductase distinct from the nitrite reductase. However, the entity of the NO reductase remains undefined.

207

The reconstituted system for the NO 2 reduction which we developed before [16] was a very good tool for the study of the mechanism of N20 formation from NO~-. Stoichiometric formation of N20 from N O ; occurred only in the presence of a sufficient quantity of the cytochrome bc 1 complex. The NO reductase, which was definitely distinct from the nitrite reductase, actually existed in the preparations of the complex. The cytochrome bc 1 complex was shown to be involved in the electron transfer for NO reduction. The major product of the purified nitrite reductase was NO, and the enzyme itself had no NO reductase activity. From these results the mechanism of N20 formation from NO2 in this photodenitrifier can be postulated as follows: first, NO~- is reduced to NO by nitrite reductase with electrons from the cytochrome bc 1 complex, and then NO is reduced immediately to N20 without release into the gas phase by the NO reductase, which is closely associated with the complex and receives electrons from it. Another question has arisen, namely whether the cytochrome bcl complex itself can be the NO reductase or whether peptide(s) with NO reductase activity other than subunits of the complex exist. Since our preparations of the complex have seven main bands and minor peptide bands on SDSpolyacrylamide gel electrophoresis, the answer has not yet been found. However, since NO reductase appears to be an inducible enzyme, it might be proposed that a peptide specific for NO reduction is associated with the cytochrome bc 1 complex.

Acknowledgement We wish to thank Dr. Keizo Shimada for helpful suggestions and discussions.

References 1 Garber, E.A.E. and Hollocher, T.C. (1981) J. Biol. Chem. 256, 5459-5465

2 Payne, W.J. (1973) Bacteriol. Rev. 37, 409-452 3 Payne, W.J. (1981) in Denitrification, pp. 79-89, John Wiley & Sons, New York 4 Payne, W.J. and Balderston, W.L. (1978) in Microbiology 1978, (Schlesinger, D. ed.) pp. 339-342, American Society for Microbiology, Washington, D.C. 5 St. John, R.T. and Hollocher, T.C. (1977) J. Biol. Chem. 252, 212-218 6 Betlach, M.R. and Tiedje, J.M. (1981) Appl. Environ. Microbiol. 42, 1074-1084 7 Wharton, D.C. and Weintraub, S.T. (1980) Biochem. Biophys. Res. Commun. 97, 236-242 8 Cox, C.D., Jr., Payne, W.J. and Dervartanian, D.V. (1971) Biochim. Biophys. Acta 253, 290-294 9 Matsubara, T. and lwasaki, H. (1971) J. Biochem. 69, 859-868 10 Sawada, E., Satoh, T. and Kitamura, H. (1978) Plant Cell Physiol. 19, 1339-1351 11 Yamanaka, T., Ota, A. and Okunuki, K. (1961) Biochim. Biophys. Acta 53, 294-308 12 Masuko, M. and Iwasaki, H. (1984) Plant Cell Physiol. 25, 439-446 13 Cox, C.D., Jr. and Payne, W.J. (1973) Can. J. Microbiol. 19, 861-872 14 Averill, B.A. and Tiedje, J.M. (1982) FEBS Lett. 138, 8-12 15 Satoh, T., Hoshino, Y. and Kitamura, H. (1976) Arch. Microbiol. 108, 265-269 16 Urata, K. and Satoh, T. (1984) FEBS Lett. 172, 205-208 17 Nicholas, D.J.D. and Nason, A. (1957) Methods Enzymol. 3, 981-984 18 Satoh, T., Horn, S.S.M. and Shanmugam, K.T. (1983) J. Bacteriol. 155, 454-458 19 Strickland, J.D.H. and Parsons, T.R. (1972) in A Practical Handbook of Seawater Analysis (Bulletin 167, 2nd Edn.), pp. 95-98, Fisheries Research Board of Canada, Ottawa 20 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 21 Hartree, E.F. (1972) Anal. Biochem. 48, 422-427 22 Hatefi, Y., Haarik, A.G. and Griffiths, D.E. (1962) J. Biol. Chem. 237, 1681-1685 23 Rich, P.R. (1981) Biochim. Biophys. Acta 637, 28-33 24 Satoh, T. (1984) Arch. Microbiol. 139, 179-183 25 Matsubara, T. (1970) J. Biochem. 67, 229-235 26 Balderston, W.L., Sherr, B. and Payne, W.J. (1976) Appl. Environ. Microbiol. 31, 504-508 27 Kristjansson, J.K. and Hollocher, T.C. (1980) J. Biol. Chem. 255, 704-707 28 Urata, K., Shimada, K. and Satoh, T. (1983) Plant Cell Physiol. 24, 501-508 29 Sawada, E. and Satoh, T. (1980) Plant Cell Physiol. 21, 205-210