RESUMEN
The action of the environmental toxic Pb(2+) on photosynthetic electron transport was studied in thylakoid membranes isolated from spinach leaves. Fluorescence and thermoluminescence techniques were performed in order to determine the mode of Pb(2+) action in photosystem II (PSII). The invariance of fluorescence characteristics of chlorophyll a (Chl a) and magnesium tetraphenylporphyrin (MgTPP), a molecule structurally analogous to Chl a, in the presence of Pb(2+) confirms that Pb cation does not interact directly with chlorophyll molecules in PSII. The results show that Pb interacts with the water oxidation complex thus perturbing charge recombination between the quinone acceptors of PSII and the S2 state of the Mn4Ca cluster. Electron transfer between the quinone acceptors QA and QB is also greatly retarded in the presence of Pb(2+). This is proposed to be owing to a transmembrane modification of the acceptor side of the photosystem.
Asunto(s)
Plomo/farmacología , Complejo de Proteína del Fotosistema II/antagonistas & inhibidores , Quinonas/antagonistas & inhibidores , Proteínas de las Membranas de los Tilacoides/antagonistas & inhibidores , Tilacoides/efectos de los fármacos , Agua/metabolismo , Clorofila/antagonistas & inhibidores , Clorofila/metabolismo , Clorofila A , Transporte de Electrón/efectos de los fármacos , Fluorescencia , Oxidación-Reducción , Fotosíntesis/efectos de los fármacos , Complejo de Proteína del Fotosistema II/metabolismo , Hojas de la Planta/química , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Quinonas/metabolismo , Spinacia oleracea/química , Spinacia oleracea/efectos de los fármacos , Spinacia oleracea/metabolismo , Proteínas de las Membranas de los Tilacoides/metabolismo , Tilacoides/química , Tilacoides/metabolismoRESUMEN
The toxic effects of Pb(2+) on photosynthetic electron transport were studied in photosystem I (PSI) submembrane fractions isolated from spinach. Structural and spectroscopic analysis using FTIR, fluorescence and X-ray photoelectron spectroscopy (XPS) showed that Pb(2+) binds with proteins via oxygen and nitrogen atoms with an overall binding constant of KPb-PSI=4.9×10(3) (±0.2) M(-1) and the number of bound Pb(2+) cation was 0.9 per PSI complex. Pb(2+) binding altered the protein conformation indicating a partial protein destabilization. Electron transport and P700 photooxidation/reduction measurements showed that the interaction of Pb(2+) cations with PSI produced a donor side limitation of electron transport presumably due to Pb(2+) binding to or in the vicinity of plastocyanin.
Asunto(s)
Cationes Bivalentes/farmacología , Plomo/farmacología , Complejo de Proteína del Fotosistema I/efectos de los fármacos , Complejo de Proteína del Fotosistema I/fisiología , Transporte de Electrón/efectos de los fármacos , Plomo/química , Complejos de Proteína Captadores de Luz/efectos de los fármacos , Complejos de Proteína Captadores de Luz/fisiología , Espectroscopía de Fotoelectrones , Fotosíntesis/efectos de los fármacos , Plastocianina/química , Conformación Proteica/efectos de los fármacos , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , Spinacia oleraceaRESUMEN
Dibromothymoquinone (DBMIB) has been used as a specific inhibitor of plastoquinol oxidation at the Q0 binding site of the cytochrome b6f complex for 40 years. It is thought to suppress electron transfer between photosystem (PS) II and I, as well as cyclic electron transfer around PSI. However, DBMIB has also been reported to act as a quencher of chlorophyll excited states. In this study, we have re-evaluated the effects of DBMIB on chlorophyll excited states and PSII photochemistry. The results show that DBMIB significantly quenches the chlorophyll excited states of PSII antenna even at low concentration (from 0.1 µM), lowering the effective excitation rate of the actinic light. It also acts as a potent PSII electron acceptor retarding the reduction of the plastoquinone pool with almost maximal potency at 2 µM. Altogether, these results suggest that experiments using DBMIB can easily be misinterpreted and stress on the importance of taking into account all these side effects that occur in the same range of DBMIB concentration used for inhibition of plastoquinol oxidation (1 µM).
Asunto(s)
Complejo de Citocromo b6f/antagonistas & inhibidores , Dibromotimoquinona/farmacología , Complejo de Proteína del Fotosistema II/metabolismo , Spinacia oleracea/efectos de los fármacos , Spinacia oleracea/metabolismo , Clorofila/metabolismo , Clorofila A , Complejo de Citocromo b6f/metabolismo , Transporte de Electrón/efectos de los fármacos , Electrones , Mediciones Luminiscentes , Simulación del Acoplamiento Molecular , Oxígeno/metabolismo , Espectrometría de Fluorescencia , Análisis Espectral , Temperatura , Tilacoides/efectos de los fármacos , Tilacoides/metabolismoRESUMEN
Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of K(Pb-HSA)â=â8.2 (±0.8)×10(4) M(-1) and K(Pb-BSA)â=â7.5 (±0.7)×10(4) M(-1). The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization.
Asunto(s)
Contaminantes Ambientales/metabolismo , Plomo/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Análisis Espectral , Animales , Sitios de Unión , Bovinos , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estabilidad ProteicaRESUMEN
The thermoluminescence afterglow (AG) measured in plant leaves originates from the S(2)/S(3)Q(B)(-) charge pair recombination in photosystem II (PSII) initiated by reverse electron flow from stromal reductants to PQ and then to the Q(B) site in PSII centers that are in the S(2)/S(3)Q(B) state. In this study, we show that this luminescence, absent in isolated thylakoid membranes, can be measured in intact chloroplasts that retain their stromal content including the electron acceptor pool (oxidized ferredoxin/NADP(+)) of photosystem I. The properties of the chloroplasts AG emission is similar to the AG in leaves in terms of temperature maximum, period-four modulation, far-red light stimulation, and antimycin A inhibition.