Systèmes Membranaires, Photobiologie, Stress et Détoxication

Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor QA to a more positive potential and lowers the yield of singlet oxygen (1O2) production. The presence of QA- results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against 1O2 in vivo under photorespiratory conditions. Here, we compared the properties of QA in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between QA and QB in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of QA to a more positive value. The yield of 1O2 production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of QA/QA- In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO2 conditions.

Membrane proteins (MPs) such as transporters, receptors, and ion channels exert key cellular functions. MPs account for one quarter of the proteins encoded by the human genome but for up to two thirds of known druggable targets, highlighting their critical pharmaceutical importance. Hence, defining the function of MPs at a molecular level and obtaining high-resolution structural information is a crucial issue; unfortunately, it has remained a difficult task to prepare and crystallize sufficient quantities of MPs for structural analysis. Heterologous expression systems have been significantly improved in the recent years, and the challenge is now to translate these improvements to crystal structures. This chapter begins with an overview of the potential of the yeast Saccharomyces cerevisiae as a host for overexpression and purification of MPs for structural purposes; a variety of already-solved structures of MPs overexpressed in S. cerevisiae are presented and the methodologies used to obtain these structures are discussed. It also examines the case of the rabbit Ca2+-ATPase Serca1a, which was expressed in S. cerevisiae in our laboratory, and describes the critical steps that led to the determination of the three-dimensional structure of this MP. Finally, expression, solubilization, stability in detergent, and functional characterization of a complex of two MPs, namely the lipid transporter (“flippase”) complex Drs2p/Cdc50p, is presented.

The bacterium Caulobacter crescentus encodes a two-component signaling protein, LovK, that contains an N-terminal photosensory LOV domain coupled to a C-terminal histidine kinase. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays regulated ATPase and autophosphorylation activity in response to visible light. Femtosecond to nanosecond visible absorption spectroscopy demonstrates congruence between full-length LovK and isolated LOV domains in the mechanism and kinetics of light-dependent cysteinyl-C4(a) adduct formation and rupture, while steady-state absorption and fluorescence line narrowing (FLN) spectroscopies reveal unique features in the electronic structure of the LovK flavin cofactor. In agreement with other sensor histidine kinases, ATP binds specifically to LovK with micromolar affinity. However, ATP binding to the histidine kinase domain of LovK has no apparent effect on global protein structure as assessed by differential Fourier transform infrared (FTIR) spectroscopy. Cysteinyl adduct formation results in only minor changes in the structure of LovK as determined by differential FTIR. This study provides insight into the structural underpinnings of LOV-mediated signal transduction in the context of a full-length histidine kinase. In particular, the data provide evidence for a model in which small changes in the tertiary/quaternary structure of LovK, as triggered by photon detection in the N-terminal LOV sensory domain, are sufficient to regulate histidine kinase activity.