Oct 18, 1980 - method developed by Buchanan and Arnon (12) and its concentration was determined spectrophotometrically at 420 nm using ee2,, = 9.7.
Communication
THEJ O U R N A L OF BIOLOGICAL CHEMISTRY Vol. %fi, N o 3, Issue of February 10, pp 1O.Z-1059, 1981 Prmted m L1.S A.
In the present communication we show that sucha conformational change does alter the affinity of the enzyme for its substrates.
Light Modulation of Chloroplast Membrane-bound FerredoxinNADP+ Oxidoreductase*
EXPERIMENTAL PROCEDURES
(Received for publication, August26, 1980, and in revised form, October 18, 1980)
Nestor Carrillo$, HectorA. Lucerog, and Ruben H.VallejosT From the Centro de Estudios Fotosinteticosy Bioquimicos, Suipacha 531,2000 Rosario-Argentina, Consejo Nacional de InvestigacionesCientificas y Tecnicas, F. M. Lillo y U. N . de Rosario, Argentina
Light increases the affinity of chloroplast membranebound ferredoxin-NADP' oxidoreductase for its physiological substrates in NADP+ photoreduction,that is, NADP+ andferredoxin.Lightmaycause this effect of the enzyme related through a conformational change to the formation of a light-drivenApH since the effect is prevented by uncouplers. Although the externalpH does influence the extent of photoreduction of NADP+ in saturating light, it does not significantly influence the extent of the energy-driven change in affinity of the enzyme forits substrates.
Chloroplasts were prepared from fresh market spinach leaves (Spinacea oleracea) as previouslydescribed (11) andsuspendedin a medium containing 250 mM sucrose, 5 mMMgC12, 10 mM TricinelNaOH (pH 8). Spinach ferredoxin was obtained and purified by the method developed by Buchanan andArnon (12) and its concentration was determined spectrophotometrically a t 420 nm using ee2,, = 9.7 (12). Chlorophyll concentration was measured as described m ~ cm." " (13). NADP' photoreduction was measured at 30°C in a reaction mixture (2.5 m l ) containing 250 mM sucrose, 5 mM MgC12, 10 mM Tricine-NaOH, pH 8, 0.5 mM NADP+, 50 p~ ferredoxin, and chloroplasts (corresponding to 10 pg of chlorophyll) unless otherwisestated. The reaction was carried out in an Aminco DW 2a spectrophotometer equipped with a magnetically stirred and thermostated3-ml cuvette. Illumination was provided by a lamp projector and the light intensity on the cuvette surface was determined by an International Light 510 A Research photometer. The photomultiplier was shielded with a filter(Corning 4-96). NADPHappearance was monitored by the increase in absorbance at 359 nm, using C:WI = 5.1 m"' cm". was measured essenDiaphorase activityof the spinach thylakoids t i d y a sdescribed in Ref. (10). NADP', Tricine. glucose 6-phosphate,and glucose-6-phosphate dehydrogenase were purchased from Sigma.All other chemicals were of analytical grade. RESULTS AND DISCUSSION
Ferredoxin-NADP+oxidoreductase (EC 1.18.1.2) is a membrane-bound component of the chloroplast photosynthetic electron transport system. Originally discovered as a specific NADPH-diaphorase by Avron and Jagendorf (1, 21, this enzyme also functionsas a pyridine nucleotide transhydrogenase (3, 4) and a NADPH-cytochrome f reductase (5, 6). The physiological role of the flavoprotein is to catalyze the final step of the photosynthetic electron transportchain, wherein ferredoxin, reduced by photosystem I in higher plants, serves as electron donorfor the flavoprotein-catalyzed reduction of NADP' ( 7 ) .Berzborn (8) and Bohme (9) have concluded that the reductase is tightly bound to thethylakoid membrane in partially protected sites andvery near to coupling factor 1. Recently we reported (10) that energization of chloroplasts by light accelerated inactivation of NADP' photoreduction by chemical modification. Uncouplers prevented the effect of light. Complete protection to theenzyme against inactivation by phenylglyoxal was afforded by 10 mM NADPf. With lower concentrations of NADP', protection was higher in the light than in the dark, suggesting that light induces a conformational change in membrane-bound ferredoxin-NADP' reductase that mayaffect the kinetic properties of the enzyme.
Effect of Light Intensity a n d Uncouplers on the Kinetic ParametersofMembrane-boundFerredoxin-NADPtReductase-Fig. lA shows theapparent X,, for NADP+when NADP+ photoreduction by spinach chloroplasts was carried out at different light intensities. There is a marked decrease in the K,,, values at high light intensities (above 6 X 10; erg. cm-2.s") compared with the values obtained from experiments carried out in dim light, revealing that the affinity of the reductase for NADP' is greater in saturating light when is fully energized. The V,, increased the thylakoid membrane with the light intensity aswas expected (data not shown). It has been shown (14) that lightcauses energization of the photosynthetic membranesby formation of a proton gradient which is strictly dependent on the light intensity. An alternative way to modify the energy state of the membranesis by decreasing the light-driven ApH by the use of uncouplers. Fig. 1B shows that the apparent K,,, for NADP' measuredin saturating light(1.5 X IO6erg.cm-2.s") increased from about 10 to 50 p ~with , increasing concentrations of NH,Cl present in the reaction medium. It was of interest, therefore, to check if the light-driven conformational change of the reductaseshould have anyeffect * This work was supported by grants from Consejo Nacional de on the affinity of the enzyme for its other physiological substrate in NADP' photoreduction, that is, ferredoxin. Fig. 2 Investigaciones Cientificas y Tecnicas (Argentina) and the Project shows that the K, for ferredoxin varies from 0.5 p~ in satuUnited Nations Development Program/United Nations Educational Scientific and Cultural Organization RLA 78/024. The costs of pubrating light and in the absence of NH&l to 4 ,UM when the lication of this article were defrayed in part by the payment of page chloroplastswerecompletelyuncoupled (more than 4 mM charges. This article must therefore be hereby marked "advertiseNH4CI). ment" in accordance with 18 U.S.C. Section 1734 solely to indicate These results indicate that (i) uncouplers completely prethis fact. vented the effect of light on the reductase and (ii) there is a $ Fellow of the Consejo Nacional de Investigaciones Cientificas y correlation between the change of affinity of the enzyme for Tecnicas (Argentina). 5 Fellow of the Consejo Nacional de Investigaciones Clentificas y its substrates inducedbylight and the ApH generated by Tecnicas (Argentina). Presentaddress, Section of Biochemistry, MO- photosynthetic electron transport. This isin good agreement lecular and Cell Biology, Cornell University, Ithaca, New York 14853. 1Career Investigatorof Consejo Nacional de Investigaciones Cientificas y Tecnicas (Argentina).
1 The abbreviation used is: Tricine, N-[tris(hydroxymethyl)methyllglycine.
1058
Light and FerredoxinNADP' Reductase TABLE I with the fact that the light effect on chemical modification of Comparison of the K,,, for NADP' in NADP' photoreduction at bound reductase is abolished by uncouplingconcentrations of different pH NH,Cl (10). Reaction mixtures (2.5 d) contained 250 m M sucrose, 5 mM MgCI2, Effect of p H on Diaphorase Activity of Thylakoid Mem20 mM Tricine-NaOH of the statedpH, 50 PM ferredoxin, chloroplasts branes-The pH profiie for the diaphorase activity of spinach (4 pgof chlorophyll/ml), and different concentrations of NADP'. chloroplasts is shown in Fig. 3. The diaphorase activity inNADP' photoreduction was measured in saturating light and K,,, creased rapidly as the pH was raised from 5.5 to 9.0. Interest- calculated as described in the text. ingly this pH activity curve is similar to those reported preK,, for NADP' pH of reaction medium viously for the soluble enzyme (1, 15), although the apparent Control 5 mM NH,CI pK for this change in the thylakoid membranes is about 7, P M that is, 1 pH unit less than the value reported (1, 15). Davis 7 35 7.0 and San Pietro (15) suggested that the pH profie indicatesa 12 50 8.0 pH-dependent transition between two different forms of the 55 11 9.0 enzyme. An ionized sulfhydryl group maybe necessaryfor the high activity form. The lower apparent pK in the membranebound enzyme seems to be another allotopic property of the photoreduction was determined a t three different pH values, ferredoxin-NADP' reductase. with or without NH,Cl in the reaction medium. The results Effect o f Light on Membrane-bound Reductase a t Different are summarized in Table I. From these data it may be conpH-In orderto distinguish therelationship between the cluded that a similar energy-dependent change in theK , for energy-driven conformat,ional change and the pH-dependent NADP' of the membrane-bound reductase did occur at any transition describedabove, the K , for NADP' in NADP+ tested pH, thus suggesting that the light effect is qualitatively different from that produced by a change in the pH of the I I I F. external medium. Since it is known that illumination of the intactchloroplast increases the stromal pH(16), the pH transition studied here C" L 1 for the membrane-bound reductase may contribute togive a more active form of the enzyme in viuo. Thus, in darkness or dim light the enzyme should exist in a rather inactive form (highK , and low activity form). When the chloroplasts are illuminated, there is an alkalinization in the stroma(which causes anincrease inthe reductase activity, without affecting the affinity for its substrates) anda conformational change generated by the ApH (lowering the K , for NADP+ and ferredoxin). These phenomena wiU greatly in40 80 120 crease the rateof NADP+ photoreduction and may play arole in the complex regulation of C02 fixation in vivo since the LIGHT INTENSITY (ERG:CM2.E.104) NHaC1 transitions to the moreefficient forms occur in conditions of FIG. 1. Effect of light intensity and NH&l on the K,,, for high light intensities when more NADPH is needed.
r-
(mil)
NADP+ of spinach ferredoxin-NADP+ reductase. Photoreduction of NADP+ by spinach thylakoids was measured as described in the text. K,, values were calculated from double reciprocal plots. In A , light intensities were as shown. In B, light intensity was saturating (1.5 X lo6 erg. ern-'. s- ') and NH,CI was added as stated. I
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