Single molecule electrochemical studies of photosynthetic complexes

Abstract

[eng] In this thesis manuscript entitled “Single molecule electrochemical studies of photosynthetic complexes” I study the Photosystem I (PSI) protein complex to characterize fundamental processes of the PSI function. PSI is a light harvesting complex located in the thylakoid membrane of algae and plants that pumps electrons throughout this membrane. Upon irradiation, PSI oxidizes plastocyanin (Pc) and reduces ferredoxin (Fd), that is, PSI is a light-driven oxidoreductase. In this dissertation, experimental results are presented regarding: i) The protein electron transfer and protein electron transport within the complex ii) The interprotein electron transfer with plastocyanin iii) The binding with plastocyanin and the influence of photosystem and plastocyanin redox state on their association iv) The excitonic-energy transfer within the light harvesting antenna of the photosystem. The novelty of the results presented here lies on the experimental approach. For this thesis, I have developed single protein techniques allowing to study single PSI, and single PSI-Pc pairs making use of nanoprobes techniques, electrochemical scanning tunnelling microscopy and spectroscopy (EC- STM and EC-TS) and atomic force microscopy and single molecule force spectroscopy (AFM, SMFS). This have been achieved optimizing the binding of PSI and Pc to gold electrodes and nanoprobes allowing to orient and face the interacting complexes. In particular I have studied the charge exchange distance of PSI and PSI-Pc complexes by tunnel current distance decay spectroscopy. I have observed, in line with previous results of the group, that proteins pairs are able to exchange charge up to several nanometers distance. I have also studied the conductance of the PSI-Pc complex. For both experiments, we have observed that Pc devoid of its Cu redox center is able to transfer charge at longer distances and with higher conductances with respect to the holo form incorporating Cu2+. I have also observed that the binding probability of the PSI-Pc complex is modulated by the redox state of the proteins. In collaboration with my colleagues we have developed a methodology to control orthogonally the redox state of PSI (with light) and Pc (with specific reducing agent). We have observed that the binding probability is enhanced when at least one of the complex is in an ET ready (PSI oxidized, Pc reduced) state. To study the excitonic energy transfer in PSI antennae, we have developed in collaboration with the institute of photonic sciences a novel photocurrent detected spectroscopic technique. We measure the electrochemical current signal, proportional to the population of charge separated states as the sample is exposed to pulsed-laser irradiation. The optical set-up consists on a fully collinear, action detected (photocurrent) two-dimensional spectroscopy. In addition, making use of PSI functionalized electrodes, I have explored the feasibility of a photocurrent-based herbicide biosensor. The photocurrent output and kinetics were systematically studied and modelled.

Altogether, I hope that the works presented in this manuscript contribute to the understanding of the electron transfer in proteins and in particular for the photosystem I complex.

[spa] En esta tesis estudio la transferencia de electrones entre proteínas usando como modelos el fotosistema I y su pareja donadora de electrones en la fotosíntesis, la plastocianina. He medido dos etapas del proceso de transferencia electrónica, la asociación/disociación del complejo que forman y el intercambio de electrones entre el par. He caracterizado estos procesos en proteínas individuales empleando nano-microscopia y espectroscopía de sonda próxima, en concreto microscopía de fuerza atómica para el estudio de la asociación/disociación y microscopía scanner de efecto túnel en medio electroquímico en los experimentos de intercambio electrónico. Para ambos estudios, he expresado mutantes de plastocianina y he desarrollado péptidos de anclaje al fotosistema I para orientar al par de proteínas sobre electrodos de oro.

Los experimentos de espectroscopía de fuerza revelan que la probabilidad de asociación entre el par de proteínas está modulada por su estado redox. En concreto, he observado que, si al menos una de las dos proteínas esté preparada para intercambiar carga, es decir si el fotosistema está oxidado o la plastocianina reducida, la probabilidad de asociación es mayor que la que observamos en el sistema fotosistema reducido y plastocianina oxidada. En el caso del intercambio electrónico, he estudiado el fotosistema I aislado, así como el par fotosistema plastocianina. En ambos casos observamos que el fotosistema es capaz de intercambiar electrones a varios nanómetros de distancia a través del electrolito con la sonda desnuda y con la sonda decorada con plastocianina respectivamente. También he estudiado el efecto del centro redox de cobre de la plastocianina en la conductancia y distancia de intercambio electrónico. Paralelamente, he explotado la fotocorriente que produce el fotosistema en dos pruebas de concepto, de carácter fundamental y aplicado, aprovechando las herramientas empleadas para medir la transferencia electrónica en el fotosistema I. Por un lado, hemos desarrollado junto con el Instituto de Ciencias Fotónicas y la universidad de Padova, un sistema de detección de fotocorriente electroquímica como observable en experimentos de espectroscopía electrónica de dos dimensiones. En la vertiente aplicada, hemos empleado la fotocorriente para determinar la concentración de un herbicida (paraquat).