Effects of hydrostatic pressure on photosynthetic activities of thylakoids

R. Hayashi and C. Balny (Editors), High Pressure Bioscience and Biotechnology 9 1996 Elsevier Science B.V. All rights reserved.

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Effects of hydrostatic pressure on photosynthetic activities of thylakoids Mitsuyoshi Yuasa a Photosynthesis Research Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan aAdvanced Research Laboratory, Hitachi Ltd., Hotoyama, Saitama 350-03, Japan

Abstract Effects of hydrostatic pressure on PS II isolated from spinach thylakoids were studied. Upon pressure treatment above 100 MPa, Mn-cluster of the oxygen-evolving enzyme was preferentially inactivated. This inactivation was effectively suppressed by inclusion of high concentration of sucrose in the medium. This enabled us to study the effects of ambient pressure on the electron transfer around PS II reaction center up to 200 MPa. Ambient high pressure retarded the electron transfer between QA and QB. It also retarded the charge recombination of S2QA- charge pair, but not that of Z§

- charge pair.

1. I N T R O D U C T I O N Light-driven electron transfer in photosynthetic membranes takes place in photosystems in which various electron donor and acceptor molecules are orderly arranged in a functional integrity consisting of more than twenty membrane proteins. As a means to understand the mechanism of electron transfer in such a semi-solid system, the pressure effect has been considered to be a method of certain interest. There have been several reports on the effects of pressure on photosynthetic electron transfer. Hydrostatic pressure affected the rates of charge separation and recombination in reaction centers of photosynthetic bacteria [ 1-4]. In intact cyanobacterial cells, pressure affected the transfer of excitation energy by inducing irreversible dissociation of protein components [5]. In view of the fact that photosystems have a supramolecular structure maintained by relatively weak intermolecular forces, the pressure effect is expected to appear in two types, reversible and irreversible. This has made it difficult to correctly examine the pressure dependence of electron transfer rates, unless both types of effects are successfully separated. In this communication, we report several features of pressure-induced irreversible damage on the oxygen-evolving enzyme of PS II, and some preliminary results on reversible effects of ambient pressure on the electron transfer in PS II reaction centers, which were enabled by use of a pressure protectant to suppress the irreversible damage.

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2. MATERIALS AND METHODS Oxygen-evolving PS II membranes were prepared from spinach basically according to the method of Berthold et al. [6]. The PS II membranes were resuspended in 0.4 M sucrose, 40 mM MES-NaOH (pH 6.5) and 20 mM NaC1. Sucrose concentration was increased to 1.2 M if needed. Cross-linked PS II membranes were prepared with EDC (1-ethyl-3-(3dimethylaminopropyl)-carbodiimide) as described by Enami et al. [7]. NHzOH treatment of PS II membranes was done as described by Ono et al. [8]. A sample contained in an inner capsule was subjected to high-pressure treatment in a highpressure cell (Hikari High Pressure Co. Ltd., Hiroshima, Japan) having three sapphire optical windows. Two different types of inner capsules were used. One was a cylindrical cell made of polyethylene tubing used for pressure treatment of samples. The pressure was increased at a constant rate to an indicated level in 3 min, kept at this level for 5 min and then released in 3 min. The treated sample (1.0 mg Chl /ml) was recovered and subjected to activity measurements. The other inner capsule was a homemade one used for measurements of fluorescence kinetics under ambient high pressure. A small piece of filter paper soaked with an aliquot of sample (2.0 mg Chl/ml, supplemented with 1.2 M sucrose) was inserted into an envelope of transparent plastic film. After sealing the opening, the envelope was placed in the high-pressure cell in a close front of the optical window. Electron transfer around the PS II reaction center was induced by a short Xe flash (10 ~ts), and was monitored by fluorescence using a pulse-modulated fluorometer (PAM system, Walz, Effeltrich, Germany).

3. RESULTS AND DISCUSSION 3.1. Pressure-induced damage of the oxygen-evolving enzyme We have recently reported that the oxygen-evolving enzyme of PS II is preferentially and selectively damaged by high-pressure treatments, while other photochemical activities of both P S I and PS II are not much affected [9]. As Fig. 1 shows, both oxygen evolution and photoreduction of DCIP (2,6-dichlorophenol-indophenol) with water as electron donor became affected by pressure treatments above 100 MPa, and inactivated almost completely at 300 MPa (0 ~ or 200 MPa (23 ~ By contrast, DCIP photoreduction with DPC (1,5diphenylcarbazide) as electron donor was stimulated. These results indicate that the oxygenevolving enzyme is specifically inactivated by high-pressure treatments, while the electron transfer from Z (the secondary donor of PS II) to QB (the secondary acceptor quinone of PS II) is resistant. It is well known that tetranuclear Mn-cluster is the molecular entity of the catalyst for water oxidation in PS II [ 10]. Inactivation of oxygen evolution, therefore, is expected to involve destruction of the Mn-cluster. Table 1 shows the changes in relative intensity of the EPR signal arising from free Mn 2+ after pressure treatment of PS II membranes. Native PS II membranes exhibited no Mn signal after washing with any buffers, whereas a large part of Mn was released from pressure-treated membranes, exhibiting strong EPR signals after washing with a buffer containing EDTA. Notably, significant amount of free Mn could be released after washing with a buffer containing no EDTA. This implies that the pressure treatments not only damaged the function of the Mn-cluster but also destroyed its structure, but most of the resultant Mn atoms remained nonspecifically adsorbed on PS II proteins in an EPR silent state.

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Fig. 1 Effects of pressure on oxygen evolution and DCIP photoreduction in normal and EDC-cross-linked PS II membranes. (A) Oxygen evolution by normal PS II after pressure treatment at 0 ~ ( 9 and 23 ~ ( I ) , and by cross-linked PS II after treatment at 0 ~ (A). (B) DCIP photoreduction by PS II membranes in the absence (solid symbols) of DPC after pressure treatment at 0 ~ ( 9 and 23 ~ (1), and in the presence (open symbols) of DPC after pressure treatment at 0 ~ ( o ) and 23 ~ (n). Open and solid triangles indicate the activity of EDC-cross-linked PS II membranes with and without DPC after pressure treatment at 0 ~ [Reprinted, with permission, from: Yuasa et al. (1995) Plant CellPhysiol.,36: 1081-1088, 9 The Japanese Society of Plant Physiologists]

~. 100 i v

8

so

c7

o 150

g 100~ '

o

~t -

50

e~ u a

O

0

100

300

400

Treatment pressure

200

( MPa )

500

Table I Release of Mn from pressure-treated PSII membranes upon washing with and without EDTA. Pressure treatments (0 ~ 5 min)

Release of Mn (%)a No wash

No treatment 300 MPa 500 MPa

0 23 33

Wash with buffer b 0 67 79

Wash with 1 mM EDTA c 0 81 90

Amount of Mn 2+ ions was estimated from the EPR signal of hyperfine lines characteristic of free Mn 2+ ions. b The buffer used was 0.4 M sucrose, 40 mM MES-NaOH (pH 6.5), 20 mM NaC1. c 1 mM EDTA was included in the sample buffer. a

A unit of PS II in oxygen-evolving membranes contains three extrinsic proteins in addition to more than twenty membrane protein components [ 10]. Of these three extrinsic proteins, the 33 kDa protein is known to stabilize the functional Mn-cluster [ 11 ]. We examined the effects of pressure treatments on the protein composition of PS II membranes by means of SDSPAGE [9]. It turned out that most of the 17 and 23 kDa extrinsic proteins were removed from the membrane after treatment at 200 MPa and above at 23 ~ for 5 min (Fig. 2). On the other hand, an appreciable amount of the 33 kDa protein was retained even after treatment at 500 MPa, although partial loss of the protein occurred above 200 MPa. These results suggest that the 33 kDa extrinsic protein no more functioned as a stabilizer of the Mn-cluster at high pressure. Presumably, high pressure caused dissociation of the 33 kDa protein from its native

70 binding site on PS II, leading thereby to destruction of the Mn-cluster, but the protein was reassociated with PS II upon releasing the pressure. Re-association of the 33 kDa protein is likely, since this protein has a high affinity for PS II [ 12]. If these considerations are true, chemical immobilization of the extrinsic proteins will protect the Mn-cluster against pressureinduced destruction. As shown in Fig. 1, EDC-cross-linked PS II in fact exhibited significant resistance to pressure treatments: higher pressures were needed for complete inactivation. Summarizing these results and considerations, we propose a scheme of pressure-induced inactivation of the oxygen-evolving enzyme as shown in Fig. 3. The initial effect of high pressure on PS II is assumed to be dissociation of the 33 kDa protein from PS II, which then facilitates the destruction of the Mn-cluster owing to the absence of the stabilizing machinery.

Fig. 2 Protein composition of PS II membranes after pressure treatment. [Reprinted, with permission, from: Yuasa et al. (1995) Plant Cell Physiol., 36:1081-1088, 9 The Japanese Society of Plant Physiologists]

Fig. 3 A scheme of pressure-induced inactivation of PS II.

71 3.2. Effects of ambient high pressure on PS II electron transfer We recently found that inclusion of high concentration of sucrose or other polyols in the medium effectively suppresses the pressure-induced inactivation of the oxygen-evolving enzyme [to be published elsewhere]. These pressure protectants enabled us to examine the effects of ambient pressure on the electron transfer in PS II by means of fluorescence. As is well established, the yield of chlorophyll fluorescence from PS II varies depending on the redox state of the primary acceptor quinone, QA [ 13], and its transient change (Fv) can be correctly monitored by use of pulse modulation fluorescence technique [ 14] with negligible or minimized photochemistry in the reaction center. Fig. 4 schematically shows the electron transfer chain around PS II reaction center. Excitation of the reaction center chlorophyll, P680, by a flash illumination results in prompt reduction of QA to QA-. The concentration of QA- decreases through three different paths. In the absence of a herbicide (DCMU), its concentration decreases owing to the forward electron transfer from QA to QB. In the presence of DCMU, its concentration decreases through recombination of S2QA- charge pair. (Note that S 2 is one equivalent oxidized state of the Mn-cluster.) In PS II depleted of the Mncluster, its concentration decreases through recombination of Z+QA- charge pair, when DCMU is present. Fig. 5 shows the effects of ambient pressure on the decay kinetics of F v after illumination with a strong single flash. The rapidly decaying component observed at 0.1 MPa (atmospheric pressure) disappeared gradually between 50 and 100 MPa, and was almost lost at 200 MPa. The low initial F v intensity immediately after flash illumination at 200 MPa may be attributed to the decrease in fluorescence yield due to the change in dielectric constant of surroundings at high pressure [ 15]. Upon releasing the pressure, these changes were largely PS II membranes photon

~

NH2OH treatment

Mn

Z ~

(So~S ,~

: P68o ~

'

DCMU Phe ~

Z+ QA" recombination

QA

4-

J

QB Pa,~ )

$2QArecombi " nation O.1 MPa (202 MPa ~ ) 2 ms

Fig. 4 Electron transfer chain around PS II reaction center in the presence or absence of the Mn-cluster and an inhibitor, DCMU.

Fig. 5 Effects of ambient pressure on the electron transfer from QA- to QB, as monitored by fluorescence decay kinetics at 20 ~

72

A A~

1

PS II membranes + DCMU

p,

(Sz QA- recombination)

I...#

~

NHzOH-treated PS II membranes + DCMU (Z + QA- recombination)

01MPa .

0.1 MPa 100MPa 200MPa m

2s

200 m s

Fig. 6 Pressure effects on the recombination of S2Q A- (A) and Z+QA- (B) charge pairs as monitored by fluorescence decay kinetics at 20 ~ restored, indicating that the effect of ambient pressure on the electron transfer from QA- to QB is reversible. Fig. 6 shows the effects of ambient pressure on fluorescence kinetics due to S2Q A- charge recombination in DCMU-treated PS II (A), and that of Z+QA - charge recombination in NH2OH-treated PS II in the presence of DCMU (B). Obviously, S2Q Acharge recombination was reversibly retarded by ambient high pressure, whereas Z+QA recombination was immune. Based on the scheme shown in Fig. 4, these results are interpreted as indicating that the reverse electron transfer from Z to S 2 is sensitive to ambient high pressure owing probably to a pressure-induced structural modulation of the Mn-cluster.

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