Maly J., Masojidek J., Pinto V., Masci A. Sugiura M. and Pilloton R.

Transcription

1 Polyaniline mediated electron transport between the histidine tagged photosystem II and gold electrode - evidence for peroxidase activity of cytochrome b-559 Maly J., Masojidek J., Pinto V., Masci A. Sugiura M. and Pilloton R.

2 Simplified structure of photosystem II

3 The model of the space arrangement of all components immobilized on the electrode surface. I [ma] 0.4 Gradual increase of peak intensity of 0.3 polyaniline during the first 10 th scans of synthesis from sulfuric acid solution. The 0.0 first scan is different from later ones (as usually mentioned in literature) and not depicted in the graph E [V]

4 The CV scans of PSII-PANI-NTA-SAM-Gold electrode A typical PSII-PANI-NTA- SAM-Gold and PANI-NTA- SAM-Gold dark scans measured under argon atmosphere at the 60 mv.s -1 scan rate in MES buffer ph = 6.5. A quasi-reversible peak pair is observed in the presence of immobilized photosystem II. Inset: dark scan for PSII-PANI-NTA- SAM-Gold was subtracted from background i.e. PANI- NTA-SAM-Gold dark electrode scan. Data are presented as the current density I [A] 4e-3 3e-3 2e-3 1e-3 0-1e-3 I [A] 1e-4 5e-5 0-5e-5-1e E vs Ag/AgCl [V] PANI-NTA-SAM-Gold PSII-PANI-NTA- SAM-Gold E vs Ag/AgCl [V] E o = +445 mv (vs NHE) which was ascribed to reduction/oxidation process of high potential cytochrome b-559 (HP cyt b-559).

5 Influence of hydrogen peroxide on PSII-PA NTA-SAM-Gold electrode I [A] 3e-3 2e-3 1e-3 0 control sample 10 um H2O2 30 um H2O2 60 um H2O2 90 um H2O2 Cyclic voltammograms of the PSII-PANI-NTA-SAM-Gold electrode in MES buffer under dark conditions as the response to the increasing concentration of hydrogen peroxide -1e-3-2e-3 1e E [V] E ' ~ +144 mv vs NHE, (100 µm H 2 O 2 ) was ascribed to low potential cytochrome b559 (LP cyt b-559) I [A] 5e-4 0-5e-4-1e-3-2e-3-2e-3 10 um H2O2 30 um H2O2 60 um H2O2 90 um H2O E [V]

6 Influence of hydrogen peroxide on PSII-PANI- NTA-SAM-Gold electrode 5e-4 4e-4 3e-4 Michaelis-Menten and Lineweaver- Burk plot of reduction of H 2 O 2 by PSII electrode I [A] 2e-4 1e-4 0 curve fitting experimentaly measured points um H 2 O 2 1/I [A] ks = 1.78 s -1 (at E 0 = 0) K m = K cat = 5180 s curve fitting experimentally measured points /uM H 2 O 2

7 Kinetic values for other peroxidases Enzyme K m K cat (s -1 ) ref. HR electrode with direct et M n.d [21+] HRP on polyaniline with direct et M n.d. [22+] HRP on polyaniline with direct et M n.d. [23+] HRP in solution n.d. ~1000 [24+] hydroperoxidase I (HPI) M 5300 [25+] katalase-peroxidase (SynKatG) M 3500 [25+]

8 Common mechanism of hydrogen peroxide reduction by various peroxidases and superoxide dismutases Peroxidase reaction: HRP[Fe +III ] + H 2 O 2 Compound I + H 2 O Compound I + Reductant Compound II Compound II + Reductant HRP[Fe +III ] SOD reaction 2 O H+ H 2 O 2 + O 2 Cyt c superoxide dismutase activity Cyt c (Fe(III)) + O 2 - O2 + cyt c Fe(II)

9 The peroxidase activity of LP cyt b-559 prevents the acceptor side photoinhibition by consumption of accumulated electrons during reduction process of hydrogen peroxide. The superoxid dismutase like activity of HP cyt b-559 could prevent the production of singlet oxygen by donation of electrons from superoxide to P680+ during the donor side photoinhibition. Proposed mechanism of the cyt b-559 role in protection of PSII against different ROS species

10 Conclusions We have developed a new original procedure for in vitro observation of redox behaviour of isolated photosystem II (PSII) The CP43 histidine-tagged PSII particles obtained from thermophilic cyanobacteria Synechococcus elongatus were selectively imobilised on modified gold electrodes with predeposited self-assembled monolayer (SAM) of nitrilotriacetic acid (NTA) chelator and conductive polymer polyaniline (PANI) The resulted electrode has shown the direct electric contact with the cytochrome b-559 of photosystem II. The peroxidase activity of cyt b-559 was observed. The basic kinetic as well as catalytic constants of this process were derived from the CV scans Based on the experimental results, a new model of cyt b-559 function was proposed and confronted with existing ones. Here, the role of cytochrome b-559 in detoxification of different ROS species as well as the prevention of singlet oxygen production is highlighted.this model has to be further experimentally verified.