Inhibition of electron transport in photosystem II by NH2OH: Further evidence for two binding sites

Mohanram Sivaraja, G Charles Dismukes

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The reaction of hydroxylamine, a substrate analogue of the water-oxidizing complex (WOC), with spinach photosystem II (PSII) membranes has been further studied by using EPR spectroscopy to monitor the stepwise oxidation of donors and reduction of electron acceptors during successive low-temperature illuminations [Sivaraja, M., & Dismukes, G. C. (1988) Biochemistry 27, 3467-3475]. In addition to its well-known binding on the donor side of PSII, hydroxylamine binds in the dark with high affinity (KD <10 μM; 2OH/PSII) to a site that structurally interacts with the primary electron acceptor FeQA -. Binding in the dark to this acceptor site causes conversion of the normal g = 1.9 EPR signal for FeQA - to g = 2.1 on the first turnover. This light-induced signal forms with or without exogenous electron acceptors and is maximized when QA is oxidized in the dark. The original g = 1.9 form recovers upon successive turnovers as the NH2OH is consumed. The binding is blocked by addition of DCMU, which displaces the secondary electron acceptor QB, or by the presence of excess quinol and NH2OH. These results indicate that the binding site for NH2OH overlaps with or interacts with the binding site for QB. The EPR microwave power saturation of the g = 2.1 signal at 5.5 K is similar to that found for the endogenous ferrosemiquinone acceptors. These results indicate a structural change in the primary acceptor site upon binding NH2OH, with no change in oxidation state of the iron or the semiquinone. In contrast, NH2OH does not bind in the dark to PSII centers exhibiting the other major form of the primary acceptor, which exhibit the g = 1.82 EPR signal, since no change in the EPR signal is observed. We also find that the high-affinity binding of NH2OH within the WOC produces no observable EPR-active products in the dark. Following illumination, this site is characterized by loss of formation of both of the EPR signals for photooxidized manganese in the S2 state, without loss of photoreduction of the primary acceptor. We conclude that this binding site is closely associated with manganese, since there exists no blockage in the photooxidation of other donors like high-potential cytochrome b559 or signal II (160Tyr-D1 protein). A new EPR signal can be observed in both untreated and NH2OH-treated PSII membranes extending over a 1000-G line width and centered at g = 2. It forms in the presence of an exogenous quinone acceptor upon multiple turnovers at 255 K in PSII membranes, or by 200 K illumination of NH2OH-inhibited membranes. It is eliminated by DCMU and by removal of manganese upon treatment with excess NH2OH. The possible identity of the species responsible for this new signal is discussed.

Original languageEnglish
Pages (from-to)6297-6306
Number of pages10
JournalBiochemistry
Volume27
Issue number17
Publication statusPublished - 1988

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Photosystem II Protein Complex
Electron Transport
Paramagnetic resonance
Binding Sites
Manganese
Lighting
Electrons
Diuron
Hydroxylamine
Membranes
Hydroquinones
Spinacia oleracea
Water
Microwaves
Biochemistry
Oxidation
Photooxidation
Spectrum Analysis
Binding sites
Iron

ASJC Scopus subject areas

  • Biochemistry

Cite this

Inhibition of electron transport in photosystem II by NH2OH : Further evidence for two binding sites. / Sivaraja, Mohanram; Dismukes, G Charles.

In: Biochemistry, Vol. 27, No. 17, 1988, p. 6297-6306.

Research output: Contribution to journalArticle

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N2 - The reaction of hydroxylamine, a substrate analogue of the water-oxidizing complex (WOC), with spinach photosystem II (PSII) membranes has been further studied by using EPR spectroscopy to monitor the stepwise oxidation of donors and reduction of electron acceptors during successive low-temperature illuminations [Sivaraja, M., & Dismukes, G. C. (1988) Biochemistry 27, 3467-3475]. In addition to its well-known binding on the donor side of PSII, hydroxylamine binds in the dark with high affinity (KD <10 μM; 2OH/PSII) to a site that structurally interacts with the primary electron acceptor FeQA -. Binding in the dark to this acceptor site causes conversion of the normal g = 1.9 EPR signal for FeQA - to g = 2.1 on the first turnover. This light-induced signal forms with or without exogenous electron acceptors and is maximized when QA is oxidized in the dark. The original g = 1.9 form recovers upon successive turnovers as the NH2OH is consumed. The binding is blocked by addition of DCMU, which displaces the secondary electron acceptor QB, or by the presence of excess quinol and NH2OH. These results indicate that the binding site for NH2OH overlaps with or interacts with the binding site for QB. The EPR microwave power saturation of the g = 2.1 signal at 5.5 K is similar to that found for the endogenous ferrosemiquinone acceptors. These results indicate a structural change in the primary acceptor site upon binding NH2OH, with no change in oxidation state of the iron or the semiquinone. In contrast, NH2OH does not bind in the dark to PSII centers exhibiting the other major form of the primary acceptor, which exhibit the g = 1.82 EPR signal, since no change in the EPR signal is observed. We also find that the high-affinity binding of NH2OH within the WOC produces no observable EPR-active products in the dark. Following illumination, this site is characterized by loss of formation of both of the EPR signals for photooxidized manganese in the S2 state, without loss of photoreduction of the primary acceptor. We conclude that this binding site is closely associated with manganese, since there exists no blockage in the photooxidation of other donors like high-potential cytochrome b559 or signal II (160Tyr-D1 protein). A new EPR signal can be observed in both untreated and NH2OH-treated PSII membranes extending over a 1000-G line width and centered at g = 2. It forms in the presence of an exogenous quinone acceptor upon multiple turnovers at 255 K in PSII membranes, or by 200 K illumination of NH2OH-inhibited membranes. It is eliminated by DCMU and by removal of manganese upon treatment with excess NH2OH. The possible identity of the species responsible for this new signal is discussed.

AB - The reaction of hydroxylamine, a substrate analogue of the water-oxidizing complex (WOC), with spinach photosystem II (PSII) membranes has been further studied by using EPR spectroscopy to monitor the stepwise oxidation of donors and reduction of electron acceptors during successive low-temperature illuminations [Sivaraja, M., & Dismukes, G. C. (1988) Biochemistry 27, 3467-3475]. In addition to its well-known binding on the donor side of PSII, hydroxylamine binds in the dark with high affinity (KD <10 μM; 2OH/PSII) to a site that structurally interacts with the primary electron acceptor FeQA -. Binding in the dark to this acceptor site causes conversion of the normal g = 1.9 EPR signal for FeQA - to g = 2.1 on the first turnover. This light-induced signal forms with or without exogenous electron acceptors and is maximized when QA is oxidized in the dark. The original g = 1.9 form recovers upon successive turnovers as the NH2OH is consumed. The binding is blocked by addition of DCMU, which displaces the secondary electron acceptor QB, or by the presence of excess quinol and NH2OH. These results indicate that the binding site for NH2OH overlaps with or interacts with the binding site for QB. The EPR microwave power saturation of the g = 2.1 signal at 5.5 K is similar to that found for the endogenous ferrosemiquinone acceptors. These results indicate a structural change in the primary acceptor site upon binding NH2OH, with no change in oxidation state of the iron or the semiquinone. In contrast, NH2OH does not bind in the dark to PSII centers exhibiting the other major form of the primary acceptor, which exhibit the g = 1.82 EPR signal, since no change in the EPR signal is observed. We also find that the high-affinity binding of NH2OH within the WOC produces no observable EPR-active products in the dark. Following illumination, this site is characterized by loss of formation of both of the EPR signals for photooxidized manganese in the S2 state, without loss of photoreduction of the primary acceptor. We conclude that this binding site is closely associated with manganese, since there exists no blockage in the photooxidation of other donors like high-potential cytochrome b559 or signal II (160Tyr-D1 protein). A new EPR signal can be observed in both untreated and NH2OH-treated PSII membranes extending over a 1000-G line width and centered at g = 2. It forms in the presence of an exogenous quinone acceptor upon multiple turnovers at 255 K in PSII membranes, or by 200 K illumination of NH2OH-inhibited membranes. It is eliminated by DCMU and by removal of manganese upon treatment with excess NH2OH. The possible identity of the species responsible for this new signal is discussed.

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