[46] Electron paramagnetic resonance-detectable Cu2+ in Synechococcus 6301 and 6311: aa3-Type cytochrome-c oxidase of cytoplasmic membrane

450

MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N2 FIXATION

[46]

[46] E l e c t r o n P a r a m a g n e t i c R e s o n a n c e - D e t e c t a b l e C u 2+ in Synechococcus 6301 a n d 6311: a a 3 - T y p e C y t o c h r o m e - c Oxidase of C y t o p l a s m i c M e m b r a n e

By

IAN V . FRY a n d G ~ N T E R A . PESCHEK

Membrane preparations from Synechococcus 6301 and 6311 exhibited low-temperature electron paramagnetic resonance (EPR) spectra in the g = 2.08 region characteristic of copper. The physical parameters of the power-saturation characteristics and the temperature-dependence profile demonstrated that the copper signals arose from a center in an environment identical to the aa3-type cytochrome-c oxidases reported in mammalian, yeast, and bacterial systems. Membrane purification procedures demonstrated that the oxidase was present in the cytoplasmic membrane at 10 times the level present in the thylakoid membrane. The copper was demonstrated to be fully redox active by its reducibility with physiological electron donors. Introduction In addition to higher plant type photosynthesis, cyanobacteria (bluegreen algae) have the capacity to carry out "mitochondrial" or dark-type respiration, and recent spectrophotometric and inhibitor studies have indicated the presence of an aa3-type cytochrome-c oxidase in membrane preparations from cyanobacteria~-7; the presence of aartype cytochrome oxidase could be verified by immunological cross-reaction with antisera against the Paracoccus denitrificans aa3-type cytochrome oxidase. 8-1° Cytochrome-c oxidase (EC 1.9.3.1) from several sources has been shown

J J. C. P. Matthijs, Ph.D. thesis. Vrije Universiteit Amsterdam, Amsterdam, The Netherlands, 1984. 2 j. C. P. Matthijs, Adv. Photosynth. Res. 2, 643 (1984), 3 j. p. Houchins and G. Hind, Plant Physiol. 76, 456 (1984). 4 G. A. Peschek, Biochim. Biophys. Acta 635, 470 (1981). 5 G. A. Peschek, Biochem. Biophys. Res. Commun. 98, 72 (1981). 6 G. A. Peschek, G. Schmetterer, G. Lauritsch, W. H. Nitschmann, P. F. Kienzl, and R. Muchl, Arch. Microbiol. 131, 261 (1982). 7 V. Molitor, W. Erber, and G. A. Peschek, FEBS Lett. 2114, 251 (1986). 8 G. A. Peschek, V. Molitor, M. Trnka, W. Wastyn, and W. Erber, this volume [45]. 9 V. Molitor, M. Trnka, and G. A. Peschek, Curr. Microbiol. 14, 263 (1987). l0 M. Tmka and G. A. Peschek, Biochem. Biophys. Res. Commun. 136, 235 (1986).

METHODS IN ENZYMOLOGY,VOL. 167

Copyright© 1988by AcademicPress, Inc. All fightsof reproductionin any formreserved.

[46]

EPR-DETECTABLE Cu2+: CYTOCHROME OXIDASE

451

to contain redox active Cu2+, 11-14and the growth of Synechococcus 6301 on CuZ+-deficient medium 15 has resulted in a marked decrease in the ability of membrane preparations from this organism to oxidize exogenous c-type cytochromes. 16 However, another source of redox active Cu E+ in photosynthetic organisms is plastocyanin, and although plastocyanin has been reported to be absent from several cyanobacterial species, 17 including Synechococcus 6301,18 the presence of small amounts of plastocyanin in these Synechococcus species could not be ruled out. In order to distinguish between the two CuE+-containing electron transport components, as well as determine the location of the cytochrome-c oxidase, we have characterized the EPR-detectable Cu 2+ signals arising from both scrambled membrane preparations (i.e., thylakoid plus cytoplasmic) and purified cytoplasmic membranes isolated from Synechococcus 6311 and 6301, which are believed to be independent isolates of the same species. 19The temperature-dependence and power-saturation characteristics of the EPR-detectable Cu 2+ signals are used to unequivocably demonstrate that the Cu 2+ exists in an environment identical to that in typical a a r t y p e cytochrome oxidases (EC 1.9.3. l) and is present in the cytoplasmic membrane. In addition, levels of EPR-detectable Cu 2+ are correlated with the capacity to oxidize reduced cytochrome c. The redox activity of the EPR-detectable Cu E+ is demonstrated by reduction with physiological electron donors. Materials and Methods

Synechococcus 6311 (ATCC 27145) and 6301 (strain 1402-1, Gottingen, FRG) were grown on Kratz and Myers media C and D, respectively, as described.15 Spheroplasts were prepared according to Biggins. 2° Crude or scrambled membranes were prepared by disrupting the sphero~1 H. Beinert, D. E. Griffith, D. C. Warton, and R. H. Sands, J. Biol. Chem. 237, 2337 (1962), ~2 B. M. Hoffmann, J. E. Roberts, M. Swanson, S. H. Speck, and E. Margoliash, Proc. Natl. Acad. Sci. U.S.A. 77, 1452 (1980). i3 A. Seelig, B. Ludwig, J. Seelig, and G. Schatz, Bioehim. Biophys. Aeta 636, 162 (1981). 14 j. A. Fee, M. G. Choc, K. L. Findling, R. Lorence, and T. Yoshida, Proc. Natl. Acad. Sci. U.S.A. 77, 147 (1980). 15 W. A. Kratz and J. Myers, Am. J. Bot. 42, 282 (1955). 16 p. F. Kienzl and G. A. Peschek, Plant Physiol. 69, 580 (1982). 17 G. Sandmann and P. Boger, Bioehim. Biophys. Aeta 766, 395 (1980). ~8 p. F. Kienzl and G. A. Peschek, FEBS Lett. 162, 76 (1983). ~9R. Rippka, J. Deruelles, J. B. Waterbury, M. Herdman, and R.Y. Stanier, J. Gen. Microbiol. U l , 1 (1979). 20 j. Biggins, Plant Physiol. 42, 1447 (1%7).

452

MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N 2 FIXATION

[46]

plasts either by passage through a French press (10,000 psi) under N2 or by ultrasonic oscillation (Branson sonifier, Model 350, microtip setting 3 for 20 min on ice). Unbroken cells were removed by centrifugation at 3,000 g (I0 min). Membranes were sedimented at 40,000 g (60 min), then resuspended in 10 mM Tris-HCl, pH 8.0, containing 10 mM EDTA, and sedimented again at 40,000 g (60 min). The EDTA served to effectively remove any adventitious paramagnetic ions such as Mn 2÷ and Cu 2÷. The pelleted membranes were resuspended in the same medium to approximately 50/xg chlorophyll/ml and frozen in liquid N2. Cytoplasmic and thylakoid membranes were prepared by the method of Peschek (this volume [45]). Purified membrane preparations were stored in liquid N2. Thawed membrane samples were centrifuged at 40,000 g (60 min); the pellet was resuspended in TES-HCI buffer, pH 7.0, containing 10 mM EDTA. Finally, the membranes were concentrated by centrifugation in quartz EPR tubes at 40,000 g (60 min) to give a final protein concentration of between 2 and 3 mg/ml. Mammalian cytochrome c oxidase was observed in submitochondrial particles (SMPs), prepared from beef heart mitochondria as described 2~ and resuspended in 10 mM EDTA, 10 m M T E S , pH 7.0. Plastocyanin was observed by EPR spectroscopy in whole cells of Gloeobacter violaceus (I. Fry, A. Robinson, and L. Packer, unpublished observations). Electron transport components in the membrane were oxidized with 50 mM potassium ferricyanide, or air, and reduced with 10 mM sodium dithionite or 10 mM ascorbate plus 25 ~ M TMPD (NN,N'N'-tetramethylp-phenylenediamine), or with 30 mM NADPH or NADH in the presence of 0.I0 mM horse heart cytochrome c and 2 mM KCN. In these experiments the exogenous cytochrome c served as an electron mediator to the cytochrome oxidase which cannot be directly reduced with NAD(P)H. H EPR spectra were recorded with a Varian E-109 spectrometer, fitted with an Air Products Heli-Trans liquid helium transfer system, at 20 mW power, 1 millitesla modulation amplitude, and 9.15 GHz. Cytochrome oxidase activity was determined from the KCN-sensitive oxidation of horse ferrocytochrome c as described previously. 6,16 Results Characterization of EPR-Detectable Cu2÷ Scrambled and purified cytoplasmic membrane preparations of Synechococcus 6301 and 6311 exhibited low-temperature EPR signals in the 2~ H. Low and L. VaUin, Biochem. Biophys. Acta 69, 361 (1963).

[46]

EPR-DETECTABLE Cu2+: CYTOCHROMEOXIDASE I

I

I I 2.5 2.4

I

I

I

!

I

I 2.3

I 2.2

I 2.1

I 2.0

I 1.0

453

g value FIG. 1. Low-temperature EPR spectra of purified cytoplasmic membranes. Conditions: 20 mW power, 1 mT modulation amplitude, 9.15 GHz, 100 K. (A) Membranes prepared as indicated in the text; (B) membranes plus 2 mM KCN; (C) membranes plus 2 mM KCN, 30 mM NADPH, 0.1 mM cytochrome c (gain used in C is 3 times that used in A and B). Substitution of N A D H for N A D P H , or incubation of the membranes with 10 mM dithionite or 10 mM acorbate plus 25/zM TMPD, gave essentially the same result as in C.

g = 2.08 region characteristic of Cu 2÷ (Fig. 1). SMPs from beef heart and whole cells of Gloeobacter oiolaceus also exhibited Cu 2÷ EPR signals which were identical in line shape to those of the Synechococcus membrane preparations (data not shown; cf. Refs. 11-13 and 22-24). The temperature-dependence profiles of the respective Cu 2÷ signals, however, clearly demonstrated that they arose from different environments (Fig. 2). The temperature dependence of the Cu 2÷ signals arising from cytoplasmic and scrambled Synechococcus membranes, and from SMPs, showed a maximum at 100 K (Fig. 1), whereas the EPR-detectable Cu 2÷ signal arising from Gloeobacter oiolaceus plastocyanin had a temperature optimum around 10-20 K (Fig. 2). 23,24 22 I. V. Fry, G. A. Peschek, M. Huttejt, and L. Packer, Biochem. Biophys. Res. Commun. 129, 109 (1985). 23 G. Sandmann and P. B6ger, Plant Sci. Lett. 17, 417 (1980). 24 H. Bohner, H. Merkle, P. Kroneck, and P. B6ger, Eur. J. Biochem. 105, 603 (1980).

454

N2 FIXATION

MEMBRANES, PIGMENTS, REDOX REACTIONS, AND

~G •=e -

i

Ioeobacter

6

/ ~

5

""

4

i

i

~~

E

c 2 CO + C~.~ 1 0

I

t~ J Synechococcus

~

e-

[46]

I;

q

/~

mitochondria

I

I

I

I

I

25

50

75

100

125

150

Temperature K FIG. 2. Temperature-dependence profile of the g = 2.08 EPR-detectable Cu 2÷ signals from S y n e c h o c o c c u s (scrambled or cytoplasmic) membranes, mitochondrial SMPs, and whole cells of G l o e o b a c t e r v i o l a c e u s . Conditions as in Fig. 1.

In addition, mixed populations of Cu 2÷ could be observed in the mitochondrial SMP preparation, giving a biphasic character to the temperature profile (Fig. 3). Treatment of SMPs with repeated freeze-thaw cycles resulted in a decrease in the Cu 2÷ signal observed at higher temperatures (100 K) with a concomitant increase in the Cu 2÷ signal observed at lower temperatures (20 K) (Figs. 3A-3C). Excessive freeze-thaw cycles (Fig. 3C) resulted in an EPR-detectable Cu 2÷ signal whose temperature-dependence profile was identical to that of cupric EDTA (data not shown). The temperature-dependence profile of the Cu 2÷ signals from Synechococcus membranes did not show any significant population of this "adventitious" or enzyme-released Cu 2÷ present in these preparations (cf. Figs. 2 and 3). The EPR Cu 2÷ signals from Synechococcus membranes and SMPs did not saturate with microwave power up to 180 mW (Fig. 4), whereas the EPR Cu 2÷ signal from Gloeobacter plastocyanin, observed at lower temperatures, readily saturated with microwave power (Fig. 4). 23,24 Addition of KCN resulted in an increase in the g = 2.08 Cu 2÷ EPR feature (Fig. 1B). Such an effect is indicative of a Cu(II)A-heme a spin interaction, which has been observed in mammalian cytochrome c oxidase. 25 This 25 G. W. Brudvig, D. F. Blair, and S. I. Chan, J . Biol. C h e m . 259, 11001 (1984).

7

I

I

I

t

I

6 A 5 4 c

3

~5 + eq o

C

1 0

0

I

I

1

I

I

25

50

75

100

125

150

Temperature K

FIG. 3. Effect of repeated freeze-thaw cycles on the temperature-dependence profile of the EPR-detectable Cu 2+ signals from mitochondrial SMPs. (A) One freeze-thaw cycle; (B) three freeze-thaw cycles; (C) five freeze-thaw cycles. Conditions as in Fig. 1.

JL $ynechococcus I

I

I

&.**

mitochondria A-

o

/~~bacter t

I

I

0

1

2

3

log P

FIG. 4. Power-saturation profile of the g = 2.08 EPR-detectable Cu 2+ signals arising from

Synechococcus (scrambled or cytoplasmic) membranes, mitochondrial SMPs, and whole cells of Gloeobacter violaceus. Conditions as in Fig. 1, except that the spectrum of Gloeobacter violaceus was measured at 20 K.

456

[46]

MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N2 FIXATION

TABLE I EFFECT OF GROWTH UNDER SALINE STRESS ON COMPONENTS OF RESPIRATORY ELECTRON TRANSPORT CHAIN Growth conditions

Whole cell endogenous respiration a

NADPH-dependent cytochrome c reduction °

Cytochromec oxidase activityc

Cu2÷ EPR g = 2.08 signala

0.015 M NaC1 0.5 M NaC1

2.95 20.65

9.5 25.5

7.8 89.8

0.66 3.58

a Expressed as p,mol O2/mg chlorophyll/hr. b Expressed as/~mol/mg chlorophyll/hr. c Reduced cytochrome c oxidation, expressed as p,mol cytochrome c oxidized/rag chlorophyll/hr. d EPR-detectable Cu2+, expressed as nmol Cu2÷/mgchlorophyll. w e a k antiferromagnetic coupling b e t w e e n the Cu 2+ and the iron centers is perturbed b y ligand binding to the heme, resulting in the o b s e r v e d increase in the Cu 2+ E P R signal.

Cytochrome Oxidase Activity and Cu 2+ Content G r o w t h o f Synechococcus 6311 under conditions of saline stress resuited in a m a r k e d increase in endogenous whole cell respiration (Table I). The increased e n d o g e n o u s respiration rate correlated well with both the c y t o c h r o m e - c oxidase activity and the Cu z÷ content of scrambled m e m b r a n e preparations. H o w e v e r , N A D P H oxidation in these preparations was not stimulated so m a r k e d l y (Table I). M e m b r a n e s p r e p a r e d by ultrasonic oscillation did not exhibit any EPR-detectable Cu 2÷ signals in the g = 2.08 region (data not shown; see Ref. 22). Characteristically, c y t o c h r o m e - c oxidase activity in sonicated or French press preparations correlated with the p r e s e n c e or a b s e n c e of EPR-detectable Cu 2÷ (data not shown; see Ref. 22).

Physiological Electron Donors and Cu 2+ Reduction C y t o p l a s m i c m e m b r a n e s w e r e incubated with N A D P H or N A D H , which h a v e b e e n shown to act as electron donors to the respiratory chain and, hence, to the c y t o c h r o m e - c oxidase of cyanobacteria, 2,26 in the presence o f K C N to p r e v e n t leakage o f electrons from the reduced oxidase. The m e m b r a n e preparations w e r e supplemented with horse heart cytoc h r o m e c to replace any soluble electron mediator(s) lost during the preparation p r o c e d u r e , z Clearly, in these experiments physiologically reduced c y t o c h r o m e c is the immediate electron d o n o r to the terminal oxidase; 26 G. A. P e s c h e k , Subcell. Biochern. 10, 83 (1984).

[46]

EPR-DETECTABLE Cu2+; CYTOCHROME OXIDASE

457

TABLE II REDOX REACTIONS OF EPR-DETECTABLE Cu 2+

Membrane type

Treatment a

Thylakoid Cytoplasmic Cytoplasmic

+ KCN + KCN +KCN, +cytochrome c, +NADPH +KCN, +cytochrome c, +NADH + KCN, +dithionite + KCN, + ascorbate, +TMPD

Cytoplasmic Cytoplasmic Cytoplasmic

EPR-detectable Cu 2+ (nmol/mg protein) 0.71 7.01 0.60 0.70 Not detectable Not detectable

a For reagent concentrations, see Methods and Materials.

horse heart ferrocytohrome c has been shown to be an excellent reductant to the membrane-bound cytochrome c oxidase of several cyanobacteria.6,7,27 The degree of reduction of the Cu 2÷ EPR signal is presented in Table II. Incubation of the cytoplasmic membrane preparation with the physiological electron donors N A D H or NADPH in the presence of cytochrome c and KCN resulted in over 90% reduction of the Cu 2÷ EPR signal (Fig. 1C, Table II), presumably by reduction to Cu ÷. Almost full reduction of the Cu 2÷ EPR signal demonstrates the integrity of complete electron transport pathway (except for the water-soluble cytochrome c lost during purification. 2 In the scrambled membrane preparations reported previously, 22 25-60% of the total EPR-detectable C u 2+ w a s not reducible by physiological electron donors. This inaccessible Cu 2÷ was attributed to damaged and nonfunctional enzyme. 28 The levels of cytochrome-c oxidase damaged by the present preparation procedures (as determined by levels of physiologically reducible Cu 2÷) were extremely low. Discussion

The membrane preparations used in this study were washed with 10 mM EDTA to remove adventitious Cu 2÷, together with other membraneassociated paramagnetic metal ions such as manganese. 29 The washing 27 G. A. Peschek, P. F. Kienzl, and G. Schmetterer, FEBS Lett. 131, 11 (1981). 28 R. Aasa, S. P. J. Albracht, K. E. Falk, B. Lanne, and T. Vanngard, Biochem. Biophys. Acta 422, 260 (1976). 29 R. Cammack, L. J. Luijk, J. J. Maguire, I. V. Fry, and L. Packer, Biochirn. Biophys. Acta 548, 267 (1979).

458

MEMBRANES, PIGMENTS, REDOX REACTIONS, AND N2 FIXATION

[46]

treatment would also remove any Cu2+-containing and other soluble proteins. Therefore, one of the very few possibilities of intrinsically membrane-bound Cu 2+ in a plastocyanin-free cyanobacterium is the aa3-type terminal oxidase, z6 which is supported by the present data. EPR spectra, temperature dependence, and power saturation of the Cu 2+ signal closely resemble EPR spectra published for the Cu2+-containing aa3-type cytochrome oxidases from mammalian mitochondria, 8,9 yeast, ~3 Paracoccus denitrificans," and Thermus thermophilus. 14 The signals are, however, clearly different from those exhibited by soluble copper proteins such as plastocyanin. 17,24,3° Moreover, both EPR-detectable Cu 2+ and cytochrome-c oxidase activity were absent in parallel from heavily sonicated membranes 22 in which the functional integrity of the cytochrome oxidase complex might have been disrupted and lost together with the Cu 2÷. In addition, a concomitant increase in both the EPR-detectable Cu 2+ and the cytochrome-c oxidase activity was observed in membrane preparations from Synechococcus sp. grown under salt stress 31 (Table I). An aa3-type cytochrome oxidase was recently described in terms of optical spectra, photoaction spectra, and differential reactivities toward various c-type cytochromes and inhibitors using membrane preparations from several axenic strains of cyanobacteria (see Ref. 24 for review). Similar findings were reported for Plectonema boryanum 1,2 and the heterocysts of Anabaena 7120. 3 In previous experiments with Synechococcus 6301 it was shown that the membrane-bound cytochrome aa3 could be physiologically reduced with horse heart ferrocytochrome c or reduced pyridine nucleotides, or with ascorbate plus TMPD or DCPIP (2,6-dichlorophenolindophenol), and oxidized with molecular oxygen4'6'16'26; now the same redox behavior has been found for the tightly membranebound Cu 2+ of the cytoplasmic membrane of Synechococcus 6311 (Table II). In scrambled membrane preparations, full reduction of the Cu 2+ by nucleotides did not occur, which may be due to a bleeding of electrons to other pathways (the oxidized chlorophyll radical was 90% reduced by nucleotides; data not shown) or due to population of Cu E+ not reducible by cytochrome c owing to damage of the cytochrome-c oxidase during membrane preparation. 28 In the purified cytoplasmic membrane preparations, however, over 90% of the EPR-detectable Cu 2+ was physiologically active, and very little "adventitious" or enzyme-released Cu 2+ was observed at low temperatures (20-30 K). The EPR signal intensity of the oxidized Cu E+ was more pronounced in the presence of KCN, owing to 3o j. W. M. Visser, J. Amsz, and B. F. Van Gelder, Biochim. Biophys. Acta 333, 279 (1974). 31 I. V. Fry, M. Huflejt, W. W. A. Erber, G. A. Peschek, and L. Packer, Arch. Biochem. Biophys. 244, 686 (1986).

[47]

N2/H2

METABOLISM; INDUCTION AND MEASUREMENT

459

perturbation of the weak Cu(II)A-heme a magnetic interaction observed in aa3-type cytochrome-c oxidases. 25 Earlier work with crude membrane fractions required Triton X-100 treatment to observe the full Cu E+ s i g n a l , 22 probably because of disruption of the magnetic interaction by the slight denaturing effect of the detergent at the concentration used. Previous results from work with intact cells, spheroplasts, or isolated membranes of Synechococcus 6301 have indicated an aa3-type terminal oxidase to be present in both cytoplasmic and thylakoid membranes. 26,32-35 Recent findings with a chlorophyll-free cytoplasmic membrane fraction from the same species showed it to be capable of oxidizing horse heart ferrocytochrome c, 7 and our present results using EPR corroborate these functional studies. From our present findings we conclude the EPR-detectable Cu E+ is present in a tightly bound form in cytoplasmic membranes of Synechococcus 6301 and 6311, at a level which is an order of magnitude greater than in thylakoid membranes. It undergoes physiological redox reactions with respiratory electron donors and acceptors, and it is identical to the aa3type cytochrome-c oxidase (EC 1.9.3.1) which has been described in mammalian and yeast mitochondria 11-13and in bacteria.13'14 32 G. A. Peschek, G. Schmetterer, G. Lauritsch, R. Muchl, P. F. Kienzl, and W. H. Nitschmann, in "Photosynthetic Prokaryotes: Cell Differentiation and Function" (G. C. Papageorgiou and L. Packer, eds.), p. 147. 1983. 33 G. A. Peschek, J. Bacteriol. 153, 539 (1983). 34 G. A. Peschek, Plant Physiol. 75, 968 (1984). 35 V. Molitor, M. Trnka, and G. A. Peschek, Curr. Microbiol. 14, 263 (1987).

[47] N i t r o g e n a n d H y d r o g e n M e t a b o l i s m : Induction and Measurement B y H E L M A R A L M O N a n d PETER BOGER

Introduction Nitrogen-fixing filamentous cyanobacteria have been studied intensively with respect to N2 and H2 gas metabolism. 1-3 This chapter summarizes some comparatively simple routine assays for the measurement of both nitrogen fixation and hydrogen gas exchange in intact cyanobacteria. l W. D. P. Stewart, Annu. Reo. Microbiol. 34, 497 (1980). 2 H. Bothe, Experientia 38, 59 (1982). 3 j. p. Houchins, Biochim. Biophys. Acta 768, 227 (1984).

METHODS IN ENZYMOLOGY, VOL. 167

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.