The energetics and evolution of oxidoreductases in deep time.

The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation-reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox (https://protein-redox-potential.web.app), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation-reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user-contributed submissions with the intention of making it a valuable resource for researchers in this field.

Using picosecond fluorescence lifetime analysis to determine photosynthesis in the world's oceans.

Phytoplankton in the ocean account for less than 1% of the global photosynthetic biomass, but contribute about 45% of the photosynthetically fixed carbon on Earth. This amazing production/biomass ratio implies a very high photosynthetic efficiency. But, how efficiently is the absorbed light used in marine photosynthesis? The introduction of picosecond and then femtosecond lasers for kinetic measurements in mid 1970s to 90 s was a revolution in basic photosynthesis research that vastly improved our understanding of the energy conversion processes in photosynthetic reactions. Until recently, the use of this technology in the ocean was not feasible due to the complexity of related instrumentation and the lack of picosecond lasers suitable for routine operation in the field. However, recent advances in solid-state laser technology and the development of compact data acquisition electronics led to the application of picosecond fluorescence lifetime analyses in the field. Here, we review the development of operational ultrasensitive picosecond fluorescence instruments to infer photosynthetic energy conversion processes in ocean ecosystems. This analysis revealed that, in spite of the high production/biomass ratio in marine phytoplankton, the photosynthetic energy conversion efficiency is exceptionally low-on average, ca. 50% of its maximum potential, suggesting that most of the contemporary open ocean surface waters are extremely nutrient deficient.

Archean phosphorus recycling facilitated by ultraviolet radiation.

Of the six elements incorporated into the major polymers of life, phosphorus is the least abundant on a global scale [E. Anders, M. Ebihara, Geochim. Cosmochim. Acta 46, 2363-2380 (1982)] and has been described as the "ultimate limiting nutrient" [T. Tyrrell, Nature 400, 525-531 (1999)]. In the modern ocean, the supply of dissolved phosphorus is predominantly sustained by the oxidative remineralization/recycling of organic phosphorus in seawater. However, in the Archean Eon (4 to 2.5 Ga), surface waters were anoxic and reducing. Here, we conducted photochemical experiments to test whether photodegradation of ubiquitous dissolved organic phosphorus could facilitate phosphorus recycling under the simulated Archean conditions. Our results strongly suggest that organic phosphorus compounds, which were produced by marine biota (e.g., adenosine monophosphate and phosphatidylserine) or delivered by meteorites (e.g., methyl phosphonate) can undergo rapid photodegradation and release inorganic phosphate into solution under anoxic conditions. Our experimental results and theoretical calculations indicate that photodegradation of organic phosphorus could have been a significant source of bioavailable phosphorus in the early ocean and would have fueled primary production during the Archean eon.

Design of a minimal di-nickel hydrogenase peptide.

Ancestral metabolic processes involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13-amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe] hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase, two ancient, extant proteins central to metabolism. These experimental results demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth.

Light-dependent signal transduction in the marine diatom Phaeodactylum tricornutum.

Unlike most higher plants, unicellular algae can acclimate to changes in irradiance on time scales of hours to a few days. The process involves an enigmatic signaling pathway originating in the plastid that leads to coordinated changes in plastid and nuclear gene expression. To deepen our understanding of this process, we conducted functional studies to examine how the model diatom, Phaeodactylum tricornutum, acclimates to low light and sought to identify the molecules responsible for the phenomenon. We show that two transformants with altered expression of two putative signal transduction molecules, a light-specific soluble kinase and a plastid transmembrane protein, that appears to be regulated by a long noncoding natural antisense transcript, arising from the opposite strand, are physiologically incapable of photoacclimation. Based on these results, we propose a working model of the retrograde feedback in the signaling and regulation of photoacclimation in a marine diatom.

Evaluating Mineral Lattices as Evolutionary Proxies for Metalloprotein Evolution.

Protein coordinated iron-sulfur clusters drive electron flow within metabolic pathways for organisms throughout the tree of life. It is not known how iron-sulfur clusters were first incorporated into proteins. Structural analogies to iron-sulfide minerals present on early Earth, suggest a connection in the evolution of both proteins and minerals. The availability of large protein and mineral crystallographic structure data sets, provides an opportunity to explore co-evolution of proteins and minerals on a large-scale using informatics approaches. However, quantitative comparisons are confounded by the infinite, repeating nature of the mineral lattice, in contrast to metal clusters in proteins, which are finite in size. We address this problem using the Niggli reduction to transform a mineral lattice to a finite, unique structure that when translated reproduces the crystal lattice. Protein and reduced mineral structures were represented as quotient graphs with the edges and nodes corresponding to bonds and atoms, respectively. We developed a graph theory-based method to calculate the maximum common connected edge subgraph (MCCES) between mineral and protein quotient graphs. MCCES can accommodate differences in structural volumes and easily allows additional chemical criteria to be considered when calculating similarity. To account for graph size differences, we use the Tversky similarity index. Using consistent criteria, we found little similarity between putative ancient iron-sulfur protein clusters and iron-sulfur mineral lattices, suggesting these metal sites are not as evolutionarily connected as once thought. We discuss possible evolutionary implications of these findings in addition to suggesting an alternative proxy, mineral surfaces, for better understanding the coevolution of the geosphere and biosphere.

Anoxic photochemical weathering of pyrite on Archean continents.

Sulfur is an essential element of life that is assimilated by Earth's biosphere through the chemical breakdown of pyrite. On the early Earth, pyrite weathering by atmospheric oxygen was severely limited, and low marine sulfate concentrations persisted for much of the Archean eon. Here, we show an anoxic photochemical mechanism of pyrite weathering that could have provided substantial amounts of sulfate to the oceans as continents formed in the late Archean. Pyrite grains suspended in anoxic ferrous iron solutions produced millimolar sulfate concentrations when irradiated with ultraviolet light. The Fe2+(aq) was photooxidized, which, in turn, led to the chemical oxidation of pyritic sulfur. Additional experiments conducted with 2.68 Ga shale demonstrated that photochemically derived ferric iron oxidizes and dissolves sedimentary pyrite during chemical weathering. The results suggest that before the rise of atmospheric oxygen, oxidative pyrite weathering on Archean continents was controlled by the exposure of land to sunlight.

Light-harvesting complex gene regulation by a MYB-family transcription factor in the marine diatom, Phaeodactylum tricornutum.

Unicellular photoautotrophs adapt to variations in light intensity by changing the abundance of light harvest pigment-protein complexes (LHCs) on time scales of hours to days. This process requires a feedback signal between the plastid (where light intensity is sensed) to the nucleus (where the genes for LHCs are encoded). The signals must include heretofore unidentified transcription factors that modify the expression level of the LHCs. Analysis of the nuclear genome of the model diatom Phaeodactylum tricornutum revealed that all the lhc genes have potential binding sites for transcription factors belonging to the MYB-family proteins. Functional studies involving antisense RNA interference of a hypothetical protein with a MYB DNA-binding domain were performed. The resultant strains with altered photosynthetic and physiological characteristics lost their ability to acclimate to changes in irradiance; i.e., cellular chlorophyll content became independent of growth irradiance. Our results strongly suggest that the inter-organellar signaling cascade was disrupted, and the cell could no longer communicate the environmental signal from the plastid to the nucleus. Here, we identify, for the first time, an LHC Regulating Myb (LRM) transcription factor, which we propose is involved in lhc gene regulation and photoacclimation mechanisms in response to changes in light intensity.

The redox state of the plastoquinone (PQ) pool is connected to thylakoid lipid saturation in a marine diatom.

The redox state of the plastoquinone (PQ) pool is a known sensor for retrograde signaling. In this paper, we asked, "does the redox state of the PQ pool modulate the saturation state of thylakoid lipids?" Data from fatty acid composition and mRNA transcript abundance analyses suggest a strong connection between these two aspects in a model marine diatom. Fatty acid profiles of Phaeodactylum tricornutum exhibited specific changes when the redox state of the PQ pool was modulated by light and two chemical inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)]. Data from liquid chromatography with tandem mass spectrometry (LC-MS/MS) indicated a ca. 7-20% decrease in the saturation state of all four conserved thylakoid lipids in response to an oxidized PQ pool. The redox signals generated from an oxidized PQ pool in plastids also increased the mRNA transcript abundance of nuclear-encoded C16 fatty acid desaturases (FADs), with peak upregulation on a timescale of 6 to 12 h. The connection between the redox state of the PQ pool and thylakoid lipid saturation suggests a heretofore unrecognized retrograde signaling pathway that couples photosynthetic electron transport and the physical state of thylakoid membrane lipids.

Quantifying structural relationships of metal-binding sites suggests origins of biological electron transfer.

Biological redox reactions drive planetary biogeochemical cycles. Using a novel, structure-guided sequence analysis of proteins, we explored the patterns of evolution of enzymes responsible for these reactions. Our analysis reveals that the folds that bind transition metal­containing ligands have similar structural geometry and amino acid sequences across the full diversity of proteins. Similarity across folds reflects the availability of key transition metals over geological time and strongly suggests that transition metal­ligand binding had a small number of common peptide origins. We observe that structures central to our similarity network come primarily from oxidoreductases, suggesting that ancestral peptides may have also facilitated electron transfer reactions. Last, our results reveal that the earliest biologically functional peptides were likely available before the assembly of fully functional protein domains over 3.8 billion years ago.Thus, life is a special, very complex form of motion of matter, but this form did not always exist, and it is not separated from inorganic nature by an impassable abyss; rather, it arose from inorganic nature as a new property in the process of evolution of the world. We must study the history of this evolution if we want to solve the problem of the origin of life. [A. I. Oparin (1)]

Using Chlorophyll Fluorescence to Determine the Fate of Photons Absorbed by Phytoplankton in the World's Oceans.

Approximately 45% of the photosynthetically fixed carbon on Earth occurs in the oceans in phytoplankton, which account for less than 1% of the world's photosynthetic biomass. This amazing empirical observation implies a very high photosynthetic energy conversion efficiency, but how efficiently is the solar energy actually used? The photon energy budget of photosynthesis can be divided into three terms: the quantum yields of photochemistry, fluorescence, and heat. Measuring two of these three processes closes the energy budget. The development of ultrasensitive, seagoing chlorophyll variable fluorescence and picosecond fluorescence lifetime instruments has allowed independent closure on the first two terms. With this closure, we can understand how phytoplankton respond to nutrient supplies on timescales of hours to months and, over longer timescales, to changes in climate.

Integrating on-grid immunogold labeling and cryo-electron tomography to reveal photosystem II structure and spatial distribution in thylakoid membranes.

A long-standing challenge in cell biology is elucidating the structure and spatial distribution of individual membrane-bound proteins, protein complexes and their interactions in their native environment. Here, we describe a workflow that combines on-grid immunogold labeling, followed by cryo-electron tomography (cryoET) imaging and structural analyses to identify and characterize the structure of photosystem II (PSII) complexes. Using an antibody specific to a core subunit of PSII, the D1 protein (uniquely found in the water splitting complex in all oxygenic photoautotrophs), we identified PSII complexes in biophysically active thylakoid membranes isolated from a model marine diatom Phaeodactylum tricornutum. Subsequent cryoET analyses of these protein complexes resolved two PSII structures: supercomplexes and dimeric cores. Our integrative approach establishes the structural signature of multimeric membrane protein complexes in their native environment and provides a pathway to elucidate their high-resolution structures.

Saturation of thylakoid-associated fatty acids facilitates bioenergetic coupling in a marine diatom allowing for thermal acclimation.

In a rapidly warming world, we ask, "What limits the potential of marine diatoms to acclimate to elevated temperatures?," a group of ecologically successful unicellular eukaryotic photoautotrophs that evolved in a cooler ocean and are critical to marine food webs. To this end, we examined thermal tolerance mechanisms related to photosynthesis in the sequenced and transformable model diatom Phaeodactylum tricornutum. Data from transmission electron microscopy (TEM) and fatty acid methyl ester-gas chromatography mass spectrometry (FAME-GCMS) suggest that saturating thylakoid-associated fatty acids allowed rapid (on the order of hours) thermal tolerance up to 28.5°C. Beyond this critical temperature, thylakoid ultrastructure became severely perturbed. Biophysical analyses revealed that electrochemical leakage through the thylakoid membranes was extremely sensitive to elevated temperature (Q10 of 3.5). Data suggest that the loss of the proton motive force (pmf) occurred even when heat-labile photosystem II (PSII) was functioning, and saturation of thylakoid-associated fatty acids was active. Indeed, growth was inhibited when leakage of pmf through thylakoid membranes was insufficiently compensated by proton input from PSII. Our findings provide a mechanistic understanding of the importance of rapid saturation of thylakoid-associated fatty acids for ultrastructure maintenance and a generation of pmf at elevated temperatures. To the extent these experimental results apply, the ability of diatoms to generate a pmf may be a sensitive parameter for thermal sensitivity diagnosis in phytoplankton.

The spatial network of skeletal proteins in a stony coral.

Coral skeletons are materials composed of inorganic aragonitic fibres and organic molecules including proteins, sugars and lipids that are highly organized to form a solid biomaterial upon which the animals live. The skeleton contains tens of proteins, all of which are encoded in the animal genome and secreted during the biomineralization process. While recent advances are revealing the functions and evolutionary history of some of these proteins, how they are spatially arranged in the skeleton is unknown. Using a combination of chemical cross-linking and high-resolution tandem mass spectrometry, we identify, for the first time, the spatial interactions of the proteins embedded within the skeleton of the stony coral Stylophora pistillata. Our subsequent network analysis revealed that several coral acid-rich proteins are invariably associated with carbonic anhydrase(s), alpha-collagen, cadherins and other calcium-binding proteins. These spatial arrangements clearly show that protein-protein interactions in coral skeletons are highly coordinated and are key to understanding the formation and persistence of coral skeletons through time.

Photosynthetic energy conversion efficiency in the West Antarctic Peninsula.

The West Antarctic Peninsula (WAP) is a highly productive polar ecosystem where phytoplankton dynamics are regulated by intense bottom-up control from light and iron availability. Rapid climate change along the WAP is driving shifts in the mixed layer depth and iron availability. Elucidating the relative role of each of these controls and their interactions is crucial for understanding of how primary productivity will change in coming decades. Using a combination of ultra-high-resolution variable chlorophyll fluorescence together with fluorescence lifetime analyses on the 2017 Palmer Long Term Ecological Research cruise, we mapped the temporal and spatial variability in phytoplankton photophysiology across the WAP. Highest photosynthetic energy conversion efficiencies and lowest fluorescence quantum yields were observed in iron replete coastal regions. Photosynthetic energy conversion efficiencies decreased by ~ 60% with a proportional increase in quantum yields of thermal dissipation and fluorescence on the outer continental shelf and slope. The combined analysis of variable fluorescence and lifetimes revealed that, in addition to the decrease in the fraction of inactive reaction centers, up to 20% of light harvesting chlorophyll-protein antenna complexes were energetically uncoupled from photosystem II reaction centers in iron-limited phytoplankton. These biophysical signatures strongly suggest severe iron limitation of photosynthesis in the surface waters along the continental slope of the WAP.

Biophysical analysis of the structural evolution of substrate specificity in RuBisCO.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant enzyme on Earth. However, its catalytic rate per molecule of protein is extremely slow and the binding of the primary substrate, CO2, is competitively displaced by O2. Hence, carbon fixation by RuBisCO is highly inefficient; indeed, in higher C3 plants, about 30% of the time the enzyme mistakes CO2 for O2 Using genomic and structural analysis, we identify regions around the catalytic site that play key roles in discriminating between CO2 and O2 Our analysis identified positively charged cavities directly around the active site, which are expanded as the enzyme evolved with higher substrate specificity. The residues that extend these cavities have recently been under selective pressure, indicating that larger charged pockets are a feature of modern RuBisCOs, enabling greater specificity for CO2 This paper identifies a key structural feature that enabled the enzyme to evolve improved CO2 sequestration in an oxygen-rich atmosphere and may guide the engineering of more efficient RuBisCOs.

Anoxic photogeochemical oxidation of manganese carbonate yields manganese oxide.

The oxidation states of manganese minerals in the geological record have been interpreted as proxies for the evolution of molecular oxygen in the Archean eon. Here we report that an Archean manganese mineral, rhodochrosite (MnCO3), can be photochemically oxidized by light under anoxic, abiotic conditions. Rhodochrosite has a calculated bandgap of about 5.4 eV, corresponding to light energy centering around 230 nm. Light at that wavelength would have been present on Earth's surface in the Archean, prior to the formation of stratospheric ozone. We show experimentally that the photooxidation of rhodochrosite in suspension with light centered at 230 nm produced H2 gas and manganite (γ-MnOOH) with an apparent quantum yield of 1.37 × 10-3 moles hydrogen per moles incident photons. Our results suggest that manganese oxides could have formed abiotically on the surface in shallow waters and on continents during the Archean eon in the absence of molecular oxygen.

Solid-State Phase Transformation and Self-Assembly of Amorphous Nanoparticles into Higher-Order Mineral Structures.

Materials science has been informed by nonclassical pathways to crystallization, based on biological processes, about the fabrication of damage-tolerant composite materials. Various biomineralizing taxa, such as stony corals, deposit metastable, magnesium-rich, amorphous calcium carbonate nanoparticles that further assemble and transform into higher-order mineral structures. Here, we examine a similar process in abiogenic conditions using synthetic, amorphous calcium magnesium carbonate nanoparticles. Applying a combination of high-resolution imaging and in situ solid-state nuclear magnetic resonance spectroscopy, we reveal the underlying mechanism of the solid-state phase transformation of these amorphous nanoparticles into crystals under aqueous conditions. These amorphous nanoparticles are covered by a hydration shell of bound water molecules. Fast chemical exchanges occur: the hydrogens present within the nanoparticles exchange with the hydrogens from the surface-bound H2O molecules which, in turn, exchange with the hydrogens of the free H2O molecule of the surrounding aqueous medium. This cascade of chemical exchanges is associated with an enhanced mobility of the ions/molecules that compose the nanoparticles which, in turn, allow for their rearrangement into crystalline domains via solid-state transformation. Concurrently, the starting amorphous nanoparticles aggregate and form ordered mineral structures through crystal growth by particle attachment. Sphere-like aggregates and spindle-shaped structures were, respectively, formed from relatively high or low weights per volume of the same starting amorphous nanoparticles. These results offer promising prospects for exerting control over such a nonclassical pathway to crystallization to design mineral structures that could not be achieved through classical ion-by-ion growth.

Small protein folds at the root of an ancient metabolic network.

Life on Earth is driven by electron transfer reactions catalyzed by a suite of enzymes that comprise the superfamily of oxidoreductases (Enzyme Classification EC1). Most modern oxidoreductases are complex in their structure and chemistry and must have evolved from a small set of ancient folds. Ancient oxidoreductases from the Archean Eon between ca. 3.5 and 2.5 billion years ago have been long extinct, making it challenging to retrace evolution by sequence-based phylogeny or ancestral sequence reconstruction. However, three-dimensional topologies of proteins change more slowly than sequences. Using comparative structure and sequence profile-profile alignments, we quantify the similarity between proximal cofactor-binding folds and show that they are derived from a common ancestor. We discovered that two recurring folds were central to the origin of metabolism: ferredoxin and Rossmann-like folds. In turn, these two folds likely shared a common ancestor that, through duplication, recruitment, and diversification, evolved to facilitate electron transfer and catalysis at a very early stage in the origin of metabolism.

How corals made rocks through the ages.

Hard, or stony, corals make rocks that can, on geological time scales, lead to the formation of massive reefs in shallow tropical and subtropical seas. In both historical and contemporary oceans, reef-building corals retain information about the marine environment in their skeletons, which is an organic-inorganic composite material. The elemental and isotopic composition of their skeletons is frequently used to reconstruct the environmental history of Earth's oceans over time, including temperature, pH, and salinity. Interpretation of this information requires knowledge of how the organisms formed their skeletons. The basic mechanism of formation of calcium carbonate skeleton in stony corals has been studied for decades. While some researchers consider coral skeletons as mainly passive recorders of ocean conditions, it has become increasingly clear that biological processes play key roles in the biomineralization mechanism. Understanding the role of the animal in living stony coral biomineralization and how it evolved has profound implications for interpreting environmental signatures in fossil corals to understand past ocean conditions. Here we review historical hypotheses and discuss the present understanding of how corals evolved and how their skeletons changed over geological time. We specifically explain how biological processes, particularly those occurring at the subcellular level, critically control the formation of calcium carbonate structures. We examine the different models that address the current debate including the tissue-skeleton interface, skeletal organic matrix, and biomineralization pathways. Finally, we consider how understanding the biological control of coral biomineralization is critical to informing future models of coral vulnerability to inevitable global change, particularly increasing ocean acidification.