Vol. 182, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS February 14, 1992 Pages 1277-l 281

Hideo Yemasaki, Shigeki Okayama*, Masatoshi Sbibata and Mitsuo Nishimura

Department of Biology, Faculty of Science, Kyushu University, BYigashi-ku, Fukuoka 812, Japan

*Biological Laboratory, College of General Education, Kyushu University, Ropponmatsu, Fukuoka 810, Japan

Received December 27, 1991

SUMMARY: The light-induced II+ release from thylakoids, which can be observed under completely uncoupled conditions, was inhibited by the SH reagent IIF ethylmaleimide (NEM) and its analogs, while the conventional H+ uptake and electron transfer were not affected. The half-inhibiting concentration of NEM for the H+ release was 10 mM and 4 mM in thylakoids in the presence of nigericin and in CFi-depleted thylakoids, respectively. The inhibitory effect increased with the increase in hydrophobicity of the NEM analogs: Z+ methylmaleimide < llFethylma.leimide < 1IFphenylmaleimide. It is suggested that SH groups in hydrophobic interior within the membrane are essential to the release of protons. 0 1992 Academic Press, Inc.

Illumination of thylakoid suspension causes H+ uptake from the

surrounding medium, a process corresponding to the vectorial H+ translocation

into the thylakoid vesicles. Removal of the catalytic moiety of the H+-ATPase

(CF1) from the membrane reverses the direction of the pH change under

illumination, namely, protons are released from thylakoids [l]. The hght-

induced H+ release is a unique phenomenon not observed in mitochondria and

bacteria [2]. The maximal amount of the H+ released is comparable to and

sometimes larger than that of the steady-state H+ uptake which can be

coupled with ATP synthesis [l]. Little is known concerning the mechanism by

which the light-response of pH change is reversed. Recently we found that

H+-conducting uncouplers and T&on-X100 also elicit the light-induced H'

release and concluded that the H+ release is a novel scalar reaction not due to

the splitting of water by PS II [2,3].

The H+ release is suppressed by inhibitors of the electron transfer [3].

However, there are no reports on specific inhibitors of the H+ release. We

report here that mthylmaleimide (NEM) and its analogs can inhibit the light-

induced H+ release from uncoupled thylakoid membranes without affecting other

photochemical activities.

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Vol. 182, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 1

Fig. 1. SDS-Gel of FKBP. Lane 1, 16/60 (2 ccg) -

FKBP b

f3 w 12

PI - 9.45 - 8.3 -7.7 I g.8

electrophoresis of the last purificationsteps Ultrogel AcA 54 (13 pg); Lane 2, Superdex 75

Fig. 2. Isoelectric focussing of FKBP. Lane 1, FKBP (Qbg); Lane 2, standard proteins.

a thirty-fold increase of the specific activity. An overall

yield of 13 % of the total activity was obtained. Figure 1

shows the SDS-PAGE of the last two purification steps. The

enzyme migrated as a single band after the Superdex

gelfiltration step and was obviously homogeneous.

Like other FKBPs from a variety of organisms the molecular

mass of this enzyme was found to lie in the range of 12 kDa.

Furthermore the enzyme exhibited peptidyl-prolyl-cis/trans

isomerase activity which could be inhibited by the

immunosuppressant FK-506 at a nanomolar level (figure 2) and

by ascomycin (not shown). Another similarity to other FKBPs

(12) lies in the preference of the enzyme for the isomerase

test-peptide succinyl-Ala-Leu-Pro-Phe-p-nitroanilide.

Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide which is a good

substrate for most cyclophilins gave only slow reaction rates

(about l/12 of that of the Leu-peptide; not shown).

Analytical isoelectrical focussing on polyacrylamid gel

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0 10 2OmM

NEM

Fig. 2. Concentration dependence of the effects of NEM on the photochemical activities of thylakoids. l , Light-induced H* release from thylakoids in the presence of 1 ILM nigericin (100% = 36 nmol H+/mg Chl). 0, Light-induced H+ release from CFi-depleted thylakoids (100% = 35 nmol H*/mg Chl). A, Light- induced H+ uptake in untreated thylakoids (100% = 290 nmol H+/mg Chl). n , Linear electron transport activity of thylakoids in the presence of 1 .uM nigericin and 100 LLM methyl viologen (100% = 81 ~mol Oz/mg Chl per h). Measurements were carried out one min after the addition of NEM in darkness. Other conditions were similar to those in Fig. 1 (A and B), except that 20 mM MOPS-KOH (pH 7.0) was added to the reaction mixture in measurements of electron flow.

nigericin. NEM abolished the flash-induced H+ release, and only the

electrochromic shift remained (Fig. 1D).

The dependence of the inhibition on NEM concentration is summarized in

Fig. 2. NEM diminished the light-induced H+ release from nigericin-added

thylakoids. In contrast, however, NEM scarcely affected the conventional

light-induced H+ uptake in untreated thylakoids and the uncoupled linear

electron flow from HzO to methyl viologen. The insensitivity of the electron

flow and H+ uptake to NEM essentially agreed with the previous results

observed at lower concentrations [4]. Decrease of the H+ uptake by NEM at

high concentrations may be due to an increase of H+ efflux via the CFc-CF1

complex [5,6].

The light-induced H+ release observed in CFl-depleted thylakoids in the

absence of nigericin was also inhibited by NEM (Fig. 2). This result

indicated that the inhibition of H+ release by NEM was not related to the CFi

complex. The depletion of CFi from the membranes reduced the concentration of

NEM for half-inhibition (10 mM in nigericin-added thylakoids and 4 mM in CFl-

depleted thylakoids at pH 7.0). It is likely that the decrease in the number

of SH groups in thylakoids reduced the half-inhibition concentration, since

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Vol. 182, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1. Inhibitory effects of maleimides on the light-induced II+ release

Reagent H+ release Inhibition (nmol H+/mg Chl) (%I

No addition 43.1 f 0.7 -

N-MM 33.3 + 0.6 23 NEM 31.3 + 0.1 27 NPM 20.9 + 0.7 52

Each reagent was added to the reaction medium at 5 mM. The H+ release was measured in the presence of 1 .uM nigericin. Other conditions were similar to those in Fig. 1 (A and B). Abbreviations used: NEM, hkthylmaleimide; NMM, N- methylmaleimide; NPM, Wphenylmaleimide.

the treatment for depletion of CFi decreases the total amount of SH groups in

the membrane by removal of peripheral proteins including the CFi complex.

The inhibitory effect depended on the hydrophobicity of the reagents

(Table 1). NMM, NEM and NPM are N-substituted maleimides, whose side chains

are methyl, ethyl and phenyl group, respectively. The inhibition increased

with the hydrophobicity of the side chain, i.e., methyl < ethyl < phenyl. The

hydrophilic thiol reagent iodoacetic acid did not inhibit the H+ release (less

than 1 % inhibition at 5 mM, not shown). The dependence of the inhibition on

reagent hydrophobicity may be explained by the difference in their

solubilities in the thylakoid membranes. The requirement for somewhat high

NEM concentrations to inhibit the H+ release can be attributed to the location

of reactive thiol groups; those thiols may be located in the hydrophobic

interior within the thylakoid membrane.

As described above, NEM inhibits the light-induced H+ release without

affecting the linear electron flow and conventional H+ uptake. The difference

in sensitivities to NEM (Fig. 2) supports our earlier suggestion that the H+

release is not a reversed H+ translocation nor a part of reactions associated

with H+ uptake [21. The H+ releasing mechanism is still obscure. Data

presented here suggest the requirement of thiol groups within the membrane for

the H+ release.

Several enzymes involved in photosynthesis are regulated by the redox

state of the enzyme thiol groups; these include the CFo-CFi complex [7],

fructose-1,6-bisphosphatase [7] and glucose++phosphate dehydrogenase [S].

Those enzymes have a Cys-X-X-X-X-X-Cys motif [g-11]. Stromal thioredoxin

regulates such enzymic activities by reducing the disulfide in vivo [7]. But

the H+ release reaction does not involve such enzymes because it can be

observed in the absence of stroma and CFi (Fig. 2).

Thylakoid membranes have the xanthophyll cycle which is believed to

dissipate excess energy of light. Violaxanthin de-epoxidase, a component of

the xanthophyll cycle, is inhibited by the SH reagent dithiothreitol and is

thought to contain a disulfide group that is necessary for activity [12,13].

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Vol. 182, No. 3, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The de-epoxidase may be a candidate for NEM-sensitive enzymes involved in the

H* release. Further investigation are in progress to identify the H+ releasing

components.

ACKNOWLEDGMENTS

We thank Dr. K. Shimazaki of Kyushu University for helpful discussions. This work was partly supported by a Grant-in-Aid for the Promotion of Science for Japanese Junior Scientists (to H. Y.) from the Ministry of Education, Science and Culture of Japan.

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