An efficient protocol for extracting thylakoid membranes and total leaf proteins from Posidonia oceanica and other polyphenol-rich plants.

BACKGROUND: The extraction of thylakoids is an essential step in studying the structure of photosynthetic complexes and several other aspects of the photosynthetic process in plants. Conventional protocols have been developed for selected land plants grown in controlled conditions. Plants accumulate defensive chemical compounds such as polyphenols to cope with environmental stresses. When the polyphenol levels are high, their oxidation and cross-linking properties prevent thylakoid extraction. RESULTS: In this study, we developed a method to counteract the hindering effects of polyphenols by modifying the grinding buffer with the addition of both vitamin C (VitC) and polyethylene glycol (PEG4000). This protocol was first applied to the marine plant Posidonia oceanica and then extended to other plants synthesizing substantial amounts of polyphenols, such as Quercus pubescens (oak) and Vitis vinifera (grapevine). Native gel analysis showed that photosynthetic complexes (PSII, PSI, and LHCII) can be extracted from purified membranes and fractionated comparably to those extracted from the model plant Arabidopsis thaliana. Moreover, total protein extraction from frozen P. oceanica leaves was also efficiently carried out using a denaturing buffer containing PEG and VitC. CONCLUSIONS: Our work shows that the use of PEG and VitC significantly improves the isolation of native thylakoids, native photosynthetic complexes, and total proteins from plants containing high amounts of polyphenols and thus enables studies on photosynthesis in various plant species grown in natural conditions.

Inter-subunit energy transfer processes in a minimal plant photosystem II supercomplex.

Photosystem II (PSII) is an integral part of the photosynthesis machinery, in which several light-harvesting complexes rely on inter-complex excitonic energy transfer (EET) processes to channel energy to the reaction center. In this paper, we report on a direct observation of the inter-complex EET in a minimal PSII supercomplex from plants, containing the trimeric light-harvesting complex II (LHCII), the monomeric light-harvesting complex CP26, and the monomeric PSII core complex. Using two-dimensional (2D) electronic spectroscopy, we measure an inter-complex EET timescale of 50 picoseconds for excitations from the LHCII-CP26 peripheral antenna to the PSII core. The 2D electronic spectra also reveal that the transfer timescale is nearly constant over the pump spectrum of 600 to 700 nanometers. Structure-based calculations reveal the contribution of each antenna complex to the measured inter-complex EET time. These results provide a step in elucidating the full inter-complex energy transfer network of the PSII machinery.

Ultrafast Excitation Energy Transfer Dynamics in the LHCII-CP29-CP24 Subdomain of Plant Photosystem II.

We measure the two-dimensional electronic spectra of the LHCII(M)-CP29-CP24 complex in photosystem II (PSII) and provide the first study of the ultrafast excitation energy transfer (EET) processes of an asymmetric and native light-harvesting assembly of the antenna of PSII. With comparisons to LHCII, we observe faster energy equilibrations in the intermediate levels of the LHCII(M)-CP29-CP24 complex at 662 and 670 nm. Notably, the putative "bottleneck" states in LHCII exhibit faster effective dynamics in the LHCII(M)-CP24-CP29 complex, with the average lifetime shortening from 2.5 ps in LHCII to 1.2 ps in the bigger assembly. The observations are supported by high-level structure-based calculations, and the accelerated dynamics can be attributed to the structural change of LHCII(M) in the bigger complex. This study shows that the biological functioning structures of the complexes are important to understand the overall EET dynamics of the PSII supercomplex.

Two-dimensional electronic spectroscopy of the Qx to Qy relaxation of chlorophylls a in photosystem II core complex.

Using two-dimensional electronic spectroscopy, we measured the Qx to Qy transfer dynamics of the chlorophyll a (Chl a) manifold in the photosystem II (PSII) monomeric core complex from Arabidopsis thaliana. A PSII monomeric core consists of 35 Chls a and no Chl b, thus allowing for a clear window to study Chl a Qx dynamics in a large pigment-protein complex. Initial excitation in the Qx band results in a transfer to the Qy band in less than 60 fs. Upon the ultrafast transfer, regardless of the excitation frequency within the Qx band, the quasi-transient absorption spectra are very similar. This observation indicates that Chl a's Qx to Qy transfer is not frequency selective. Using a simple model, we determined that this is not due to the lifetime broadening of the ultrafast transfer but predominantly due to a lack of correlation between the PSII core complex's Chl a Qx and Qy bands. We suggest the origin to be the intrinsic loss of correlation during the Qx to Qy internal conversion as observed in previous studies of molecular Chl a dissolved in solvents.

A guanosine tetraphosphate (ppGpp) mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis.

Guanosine pentaphosphate and tetraphosphate (together referred to as ppGpp) are hyperphosphorylated nucleotides found in bacteria and the chloroplasts of plants and algae. In plants and algae artificial ppGpp accumulation can inhibit chloroplast gene expression, and influence photosynthesis, nutrient remobilization, growth, and immunity. However, it is so far unknown whether ppGpp is required for abiotic stress acclimation in plants. Here, we demonstrate that ppGpp biosynthesis is necessary for acclimation to nitrogen starvation in Arabidopsis. We show that ppGpp is required for remodeling the photosynthetic electron transport chain to downregulate photosynthetic activity and for protection against oxidative stress. Furthermore, we demonstrate that ppGpp is required for coupling chloroplastic and nuclear gene expression during nitrogen starvation. Altogether, our work indicates that ppGpp is a pivotal regulator of chloroplast activity for stress acclimation in plants.

Plastoquinone homeostasis in plant acclimation to light intensity.

Arabidopsis plants were grown from seeds at different photon flux densities (PFDs) of white light ranging from 65 to 800 µmol photons m-2 s-1. Increasing PFD brought about a marked accumulation of plastoquinone (PQ) in leaves. However, the thylakoid photoactive PQ pool, estimated to about 700 pmol mg-1 leaf dry weight, was independent of PFD; PQ accumulation in high light mostly occurred in the photochemically non-active pool (plastoglobules, chloroplast envelopes) which represented up to 75% of total PQ. The amounts of PSII reaction center (on a leaf dry weight basis) also were little affected by PFD during growth, leading to a constant PQ/PSII ratio at all PFDs. Boosting PQ biosynthesis by overexpression of a solanesyl diphosphate-synthesizing enzyme strongly enhanced the PQ levels, particularly at high PFDs. Again, this accumulation occurred exclusively in the non-photoactive PQ pool. Mutational suppression of the plastoglobular ABC1K1 kinase led to a selective reduction of the thylakoid PQ pool size to ca. 400 pmol mg-1 in a large range of PFDs, which was associated with a restriction of the photosynthetic electron flow. Our results show that photosynthetic acclimation to light intensity does not involve modulation of the thylakoid PQ pool size or the amounts of PSII reaction centers. There appears to be a fixed amount of PQ molecules for optimal interaction with PSII and efficient photosynthesis, with the extra PQ molecules being stored outside the thylakoid membranes, implying a tight regulation of PQ distribution within the chloroplasts.

Precise estimation of chlorophyll a, b and carotenoid content by deconvolution of the absorption spectrum and new simultaneous equations for Chl determination.

The precise determination of photosynthetic pigment content in green organisms, chlorophylls (Chls) and carotenoids (Cars), is important to investigate many photosynthetic processes such as responses to environmental fluctuations or to gene mutations, as well as to interpret biochemical and structural results obtained on purified membranes and photosynthetic complexes. The most utilized methods for determination by spectrophotometry of Chl content in solution, usually 80% acetone, are based on the use of simultaneous equations. The advantages are the easiness and speed over chromatography, which also requires less common equipment. The disadvantage is that issues in sample preparation or in the measurement are not detectable, which could lead to wrong results. Here we propose a fast, accurate and (almost) error-proof method to measure Chl a, Chl b and also total Car content in a solution of pigments extracted from tissue, membranes or purified complexes. The method is based on the fit of the absorption spectrum of the acetone extract using the spectra of purified pigments as references. We show how this method allows a more precise and accurate estimation of pigment content as compared to classical equations, even in incorrectly prepared acetone solutions. Moreover, the method allows the discovery of artifacts in sample preparation or measurement and thus drastically reduces the risk of mistakes. Examples obtained on purified complexes are also discussed. Based on newly acquired Chl spectra, we also propose a new set of improved simultaneous equations that provide slightly different but more reliable results in comparison with the currently used equations.

PsbS contributes to photoprotection in Chlamydomonas reinhardtii independently of energy dissipation.

Photosynthetic organisms are frequently exposed to excess light conditions and hence to photo-oxidative stress. To counteract photo-oxidative damage, land plants and most algae make use of non- photochemical quenching (NPQ) of excess light energy, in particular the rapidly inducible and relaxing qE-mechanism. In vascular plants, the constitutively active PsbS protein is the key regulator of qE. In the green algae C. reinhardtii, however, qE activation is only possible after initial high-light (HL) acclimation for several hours and requires the synthesis of LHCSR proteins which act as qE regulators. The precise function of PsbS, which is transiently expressed during HL acclimation in C. reinhardtii, is still unclear. Here, we investigated the impact of different PsbS amounts on HL acclimation characteristics of C. reinhardtii cells. We demonstrate that lower PsbS amounts negatively affect HL acclimation at different levels, including NPQ capacity, electron transport characteristics, antenna organization and morphological changes, resulting in an overall increased HL sensitivity and lower vitality of cells. Contrarily, higher PsbS amounts do not result in a higher NPQ capacity, but nevertheless provide higher fitness and tolerance towards HL stress. Strikingly, constitutively expressed PsbS protein was found to be degraded during HL acclimation. We propose that PsbS is transiently required during HL acclimation for the reorganization of thylakoid membranes and/or antenna proteins along with the activation of NPQ and adjustment of electron transfer characteristics, and that degradation of PsbS is essential in the fully HL acclimated state.

Isolation and characterization of a large photosystem I-light-harvesting complex II supercomplex with an additional Lhca1-a4 dimer in Arabidopsis.

The biological conversion of light energy into chemical energy is performed by a flexible photosynthetic machinery located in the thylakoid membranes. Photosystems I and II (PSI and PSII) are the two complexes able to harvest light. PSI is the last complex of the electron transport chain and is composed of multiple subunits: the proteins building the catalytic core complex that are well conserved between oxygenic photosynthetic organisms, and, in green organisms, the membrane light-harvesting complexes (Lhc) necessary to increase light absorption. In plants, four Lhca proteins (Lhca1-4) make up the antenna system of PSI, which can be further extended to optimize photosynthesis by reversible binding of LHCII, the main antenna complex of photosystem II. Here, we used biochemistry and electron microscopy in Arabidopsis to reveal a previously unknown supercomplex of PSI with LHCII that contains an additional Lhca1-a4 dimer bound on the PsaB-PsaI-PsaH side of the complex. This finding contradicts recent structural studies suggesting that the presence of an Lhca dimer at this position is an exclusive feature of algal PSI. We discuss the features of the additional Lhca dimer in the large plant PSI-LHCII supercomplex and the differences with the algal PSI. Our work provides further insights into the intricate structural plasticity of photosystems.

Photoprotection and growth under different lights of Arabidopsis single and double mutants for energy dissipation (npq4) and state transitions (pph1).

KEY MESSAGE: Arabidopsis single and double mutants for energy dissipation (npq4) and state transitions (pph1, blocked in State II) show enhanced growth and flowers + siliques production under controlled low-light conditions. Non-photochemical quenching (NPQ) is a short-term regulation important to maintain efficient photosynthesis and to avoid photooxidative damages by dissipation of excess energy. Full activation of NPQ in plants requires the protonation of the PsbS protein, which is the sensor of the low lumenal pH triggering the thermal dissipation. State transitions are a second important photosynthetic regulation to respond to changes in light quality and unbalanced excitation of photosystems. State transitions allow energy redistribution between PSI and PSII through the reversible exchange of LHCII antenna complexes between photosystems thanks to the opposite action of the STN7 kinase and PPH1 phosphatase: phosphorylation of LHCII promotes its mobilization from PSII to PSI, while dephosphorylation has the opposite effect. In this work, we produced the pph1/npq4 double mutant and characterized some photosynthetic, growth and reproduction properties in comparison with wild-type and single-mutant plants in high- and low-light conditions. Results indicate that in high light, the pph1 mutant maintains good photoprotection ability, while npq4 plants show more susceptibility to photodamages. The pph1/npq4 double mutant showed a resistance to high-light stress similar to that of the single npq4 mutant. In low-light condition, the single mutants showed a significant increase of growth and flowering compared to wild-type plants and this effect was further enhanced in the pph1/npq4 double mutant. Results suggest that photosynthetic optimisation to improve crop growth and productivity might be possible, at least under controlled low-light conditions, by modifying NPQ and regulation of state transitions.

Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots.

Numerous proteins require a metallic co-factor for their function. In plastids, the maturation of iron-sulfur (Fe-S) proteins necessitates a complex assembly machinery. In this study, we focused on Arabidopsis thaliana NFU1, NFU2, and NFU3, which participate in the final steps of the maturation process. According to the strong photosynthetic defects observed in high chlorophyll fluorescence 101 (hcf101), nfu2, and nfu3 plants, we determined that NFU2 and NFU3, but not NFU1, act immediately upstream of HCF101 for the maturation of [Fe4S4]-containing photosystem I subunits. An additional function of NFU2 in the maturation of the [Fe2S2] cluster of a dihydroxyacid dehydratase was obvious from the accumulation of precursors of the branched-chain amino acid synthesis pathway in roots of nfu2 plants and from the rescue of the primary root growth defect by supplying branched-chain amino acids. The absence of NFU3 in roots precluded any compensation. Overall, unlike their eukaryotic and prokaryotic counterparts, which are specific to [Fe4S4] proteins, NFU2 and NFU3 contribute to the maturation of both [Fe2S2] and [Fe4S4] proteins, either as a relay in conjunction with other proteins such as HCF101 or by directly delivering Fe-S clusters to client proteins. Considering the low number of Fe-S cluster transfer proteins relative to final acceptors, additional targets probably await identification.

Functions and Evolution of Lhcb Isoforms Composing LHCII, the Major Light Harvesting Complex of Photosystem II of Green Eukaryotic Organisms.

Oxygenic photosynthesis provides energy and oxygen for almost all forms of life on earth. This process is based on the energy of photons, which is used to split water and use its electrons to reduce carbon atoms to create organic molecules and thus fix the light energy into a chemical form. Two photosytems working in series are involved in light harvesting and conversion. Both are multi-protein supercomplexes composed of a core complex, where the photochemical reaction takes place, and an antenna system involved in light harvesting. In plants and green algae, the antenna of photosystem II, the photosynthetic complex involved in water splitting, comprises the Light Harvesting Complex II (LHCII) trimers, the most abundant membrane protein on earth. LHCII is composed of highly conserved Lhcb isoforms and all green organisms count a high number of Lhcb. In vascular plants they are classified in three distinct subclasses, Lhcb1, 2 and 3, while in algae and non-vascular plants, these isoforms are less differentiated and called Lhcbm proteins. In this review, we compare LHCII proteins of different organisms, from green algae to angiosperms, and discuss the role of the modifications that occurred through evolution. We highlight the various functions of the different isoforms in photosynthesis, ranging from light harvesting, a common role to all these proteins, to regulations of photosynthesis that rely on specific isoforms.

Subunit and chlorophyll organization of the plant photosystem II supercomplex.

Photosystem II (PSII) is a light-driven protein, involved in the primary reactions of photosynthesis. In plant photosynthetic membranes PSII forms large multisubunit supercomplexes, containing a dimeric core and up to four light-harvesting complexes (LHCs), which act as antenna proteins. Here we solved a three-dimensional (3D) structure of the C2S2M2 supercomplex from Arabidopsis thaliana using cryo-transmission electron microscopy (cryo-EM) and single-particle analysis at an overall resolution of 5.3 Å. Using a combination of homology modelling and restrained refinement against the cryo-EM map, it was possible to model atomic structures for all antenna complexes and almost all core subunits. We located all 35 chlorophylls of the core region based on the cyanobacterial PSII structure, whose positioning is highly conserved, as well as all the chlorophylls of the LHCII S and M trimers. A total of 13 and 9 chlorophylls were identified in CP26 and CP24, respectively. Energy flow from LHC complexes to the PSII reaction centre is proposed to follow preferential pathways: CP26 and CP29 directly transfer to the core using several routes for efficient transfer; the S trimer is directly connected to CP43 and the M trimer can efficiently transfer energy to the core through CP29 and the S trimer.

Kinetics and heterogeneity of energy transfer from light harvesting complex II to photosystem I in the supercomplex isolated from Arabidopsis.

State transitions are a phenomenon that maintains the excitation balance between photosystem II (PSII) and photosystem I (PSI-LHCI) by controlling their relative absorption cross-sections. Under light conditions exciting PSII preferentially, a trimeric LHCII antenna moves from PSII to PSI-LHCI to form the PSI-LHCI-LHCII supercomplex. In this work, the excited state dynamics in the PSI-LHCI and PSI-LHCI-LHCII supercomplexes isolated from Arabidopsis have been investigated by picosecond time-resolved fluorescence spectroscopy. The excited state decays were analysed using two approaches based on either (i) a sum of discrete exponentials or (ii) a continuous distribution of lifetimes. The results indicate that the energy transfer from LHCII to the bulk of the PSI antenna occurs with an average macroscopic transfer rate in the 35-65 ns-1 interval. Yet, the most satisfactory description of the data is obtained when considering a heterogeneous population containing two PSI-LHCI-LHCII supercomplexes characterised by a transfer time of ∼15 and ∼60 ns-1, likely due to the differences in the strength and orientation of LHCII harboured to PSI. Both these values are of the same order of magnitude of those estimated for the average energy transfer rates from the low energy spectral forms of LHCI to the bulk of the PSI antenna (15-40 ns-1), but they are slower than the transfer from the bulk antenna of PSI to the reaction centre (>150 ns-1), implying a relatively small kinetics bottleneck for the energy transfer from LHCII. Nevertheless, the kinetic limitation imposed by excited state diffusion has a negligible impact on the photochemical quantum efficiency of the supercomplex, which remains about 98% in the case of PSI-LHCI.

Biochemical and Spectroscopic Characterization of Highly Stable Photosystem II Supercomplexes from Arabidopsis.

Photosystem II (PSII) is a large membrane supercomplex involved in the first step of oxygenic photosynthesis. It is organized as a dimer, with each monomer consisting of more than 20 subunits as well as several cofactors, including chlorophyll and carotenoid pigments, lipids, and ions. The isolation of stable and homogeneous PSII supercomplexes from plants has been a hindrance for their deep structural and functional characterization. In recent years, purification of complexes with different antenna sizes was achieved with mild detergent solubilization of photosynthetic membranes and fractionation on a sucrose gradient, but these preparations were only stable in the cold for a few hours. In this work, we present an improved protocol to obtain plant PSII supercomplexes that are stable for several hours/days at a wide range of temperatures and can be concentrated without degradation. Biochemical and spectroscopic properties of the purified PSII are presented, as well as a study of the complex solubility in the presence of salts. We also tested the impact of a large panel of detergents on PSII stability and found that very few are able to maintain the integrity of PSII. Such new PSII preparation opens the possibility of performing experiments that require room temperature conditions and/or high protein concentrations, and thus it will allow more detailed investigations into the structure and molecular mechanisms that underlie plant PSII function.

Chlamydomonas reinhardtii PsbS Protein Is Functional and Accumulates Rapidly and Transiently under High Light.

Photosynthetic organisms must respond to excess light in order to avoid photo-oxidative stress. In plants and green algae the fastest response to high light is non-photochemical quenching (NPQ), a process that allows the safe dissipation of the excess energy as heat. This phenomenon is triggered by the low luminal pH generated by photosynthetic electron transport. In vascular plants the main sensor of the low pH is the PsbS protein, while in the green alga Chlamydomonas reinhardtii LhcSR proteins appear to be exclusively responsible for this role. Interestingly, Chlamydomonas also possesses two PsbS genes, but so far the PsbS protein has not been detected and its biological function is unknown. Here, we reinvestigated the kinetics of gene expression and PsbS and LhcSR3 accumulation in Chlamydomonas during high light stress. We found that, unlike LhcSR3, PsbS accumulates very rapidly but only transiently. In order to determine the role of PsbS in NPQ and photoprotection in Chlamydomonas, we generated transplastomic strains expressing the algal or the Arabidopsis psbS gene optimized for plastid expression. Both PsbS proteins showed the ability to increase NPQ in Chlamydomonas wild-type and npq4 (lacking LhcSR3) backgrounds, but no clear photoprotection activity was observed. Quantification of PsbS and LhcSR3 in vivo indicates that PsbS is much less abundant than LhcSR3 during high light stress. Moreover, LhcSR3, unlike PsbS, also accumulates during other stress conditions. The possible role of PsbS in photoprotection is discussed.

An Ancient Bacterial Signaling Pathway Regulates Chloroplast Function to Influence Growth and Development in Arabidopsis.

The chloroplast originated from the endosymbiosis of an ancient photosynthetic bacterium by a eukaryotic cell. Remarkably, the chloroplast has retained elements of a bacterial stress response pathway that is mediated by the signaling nucleotides guanosine penta- and tetraphosphate (ppGpp). However, an understanding of the mechanism and outcomes of ppGpp signaling in the photosynthetic eukaryotes has remained elusive. Using the model plant Arabidopsis thaliana, we show that ppGpp is a potent regulator of chloroplast gene expression in vivo that directly reduces the quantity of chloroplast transcripts and chloroplast-encoded proteins. We then go on to demonstrate that the antagonistic functions of different plant RelA SpoT homologs together modulate ppGpp levels to regulate chloroplast function and show that they are required for optimal plant growth, chloroplast volume, and chloroplast breakdown during dark-induced and developmental senescence. Therefore, our results show that ppGpp signaling is not only linked to stress responses in plants but is also an important mediator of cooperation between the chloroplast and the nucleocytoplasmic compartment during plant growth and development.

The specific localizations of phosphorylated Lhcb1 and Lhcb2 isoforms reveal the role of Lhcb2 in the formation of the PSI-LHCII supercomplex in Arabidopsis during state transitions.

State transitions are an important photosynthetic short-term response that maintains the excitation balance between photosystems I (PSI) and II (PSII). In plants, when PSII is preferentially excited, LHCII, the main heterotrimeric light harvesting complex of PSII, is phosphorylated by the STN7 kinase, detaches from PSII and moves to PSI to equilibrate the relative absorption of the two photosystems (State II). When PSI is preferentially excited LHCII is dephosphorylated by the PPH1 (TAP38) phosphatase, and returns to PSII (State I). Phosphorylation of LHCII that remain bound to PSII has also been observed. Although the kinetics of LHCII phosphorylation are well known from a qualitative standpoint, the absolute phosphorylation levels of LHCII (and its isoforms) bound to PSI and PSII have been little studied. In this work we thoroughly investigated the phosphorylation level of the Lhcb1 and Lhcb2 isoforms that compose LHCII in PSI-LHCII and PSII-LHCII supercomplexes purified from WT and state transition mutants of Arabidopsis thaliana. We found that, at most, 40% of the monomers that make up PSI-bound LHCII trimers are phosphorylated. Phosphorylation was much lower in PSII-bound LHCII trimers reaching only 15-20%. Dephosphorylation assays using a recombinant PPH1 phosphatase allowed us to investigate the role of the two isoforms during state transitions. Our results strongly suggest that a single phosphorylated Lhcb2 is sufficient for the formation of the PSI-LHCII supercomplex. These results are a step towards a refined model of the state transition phenomenon and a better understanding of the short-term response to changes in light conditions in plants.

A microfluidic flow-cell for the study of the ultrafast dynamics of biological systems.

The study of biochemical dynamics by ultrafast spectroscopic methods is often restricted by the limited amount of liquid sample available, while the high repetition rate of light sources can induce photodamage. In order to overcome these limitations, we designed a high flux, sub-ml, capillary flow-cell. While the 0.1 mm thin window of the 0.5 mm cross-section capillary ensures an optimal temporal resolution and a steady beam deviation, the cell-pump generates flows up to ∼0.35 ml/s that are suitable to pump laser repetition rates up to ∼14 kHz, assuming a focal spot-diameter of 100 µm. In addition, a decantation chamber efficiently removes bubbles and allows, via septum, for the addition of chemicals while preserving the closed atmosphere. The minimal useable amount of sample is ∼250 µl.

Spectroscopic characterization of a green copper site in a single-domain cupredoxin.

Cupredoxins are widespread copper-binding proteins, mainly involved in electron transfer pathways. They display a typical rigid greek key motif consisting of an eight stranded ß-sandwich. A fascinating feature of cupredoxins is the natural diversity of their copper center geometry. These geometry variations give rise to drastic changes in their color, such as blue, green, red or purple. Based on several spectroscopic and structural analyses, a connection between the geometry of their copper-binding site and their color has been proposed. However, little is known about the relationship between such diversity of copper center geometry in cupredoxins and possible implications for function. This has been difficult to assess, as only a few naturally occurring green and red copper sites have been described so far. We report herein the spectrocopic characterization of a novel kind of single domain cupredoxin of green color, involved in a respiratory pathway of the acidophilic organism Acidithiobacillus ferrooxidans. Biochemical and spectroscopic characterization coupled to bioinformatics analysis reveal the existence of some unusual features for this novel member of the green cupredoxin sub-family. This protein has the highest redox potential reported to date for a green-type cupredoxin. It has a constrained green copper site insensitive to pH or temperature variations. It is a green-type cupredoxin found for the first time in a respiratory pathway. These unique properties might be explained by a region of unknown function never found in other cupredoxins, and by an unusual length of the loop between the second and the fourth copper ligands. These discoveries will impact our knowledge on non-engineered green copper sites, whose involvement in respiratory chains seems more widespread than initially thought.