Biology subjects

Appel, J.

Publications and source records attributed to Appel, J..

6 recordsLinked to original sources

bioRxiv2026

Physiological basis of photosynthetic hydrogen production in the cyanobacterium Synechocystis

Photosynthetic hydrogen (photoH2) production by the cyanobacterium Synechocystis sp. PCC 6803 is an attractive means for storing solar energy. However, photoH2 yields remain limited by competing electron flux pathways. Recent in vitro characterization suggests that photoH2 production requires electrons from both carbohydrate oxidation and photosynthesis. Engineered fusions between photosystem I (PSI) and hydrogenase (PSI-H2ase) aim to divert electrons toward H2 production and rely exclusively on photosynthesis. Thus, photoH2 production differs fundamentally between wildtype (WT) and PSI-H2ase fusion mutants. Here, we show that photoH2 production in WT is enhanced by supplemented glucose, consistent with the recently reported confurcating nature of HoxEFUYH H2ases. PhotoH2 production was further studied in the new psaE-hoxUYH mutant by simultaneously monitoring electron flux through PSI alongside with turnover rates of O2, CO2 and H2. PsaE-hoxUYH achieved the highest photoH2 yield and longest production period among the currently available PSI-H2ase mutants in Synechocystis, prolonged by removing O2. Upon illumination, psaE-hoxUYH exhibited high initial photoH2 production rates, which decreased in parallel with CO2 fixation and ceased immediately in the presence of O2. In absence of O2, photoH2 production still declined slowly. Therefore, in addition to CO2 fixation and O2, other yet unknown factors might limit photoH2 production under these conditions. Moreover, we traced a previously observed high H2 production phase of unclear origin in psaD-hoxYH cultures to contaminating [FeFe]-H2ases from Clostridium intestinale rather than genuine photoH2 production by the mutant. Together, these findings indicate a complex metabolic interplay tuning photoH2 production in Synechocystis WT and PSI-H2ase fusion mutants.

microbiology↗

bioRxiv2026

Electron confurcation drives photosynthetic H2 production in cyanobacteria

Cyanobacteria are major contributors to global photosynthesis and are intensively studied for sustainable green H2 production. Central to this process is the bidirectional [NiFe]-hydrogenase HoxEFUYH, yet its physiological redox partners have remained unresolved. Ferredoxin, NAD(H), and NADP(H) have been proposed as partners, but the lack of active enzyme preparations has prevented a definitive assignment. Here, we purified the intact HoxEFUYH complex from Synechocystis sp. PCC 6803 under strictly anaerobic conditions and reveal its function as both a bifurcating and confurcating hydrogenase. During H2 uptake, HoxEFUYH utilizes NAD+ and oxidized ferredoxin, whereas H2 production strictly requires both NADH and reduced ferredoxin; NADPH does not support either reaction. Combining high-resolution cryo-electron microscopy with biochemical and spectroscopic analyses, our data reveal that an flavin-containing reductase module is electronically connected to the catalytic [NiFe]-hydrogenase core through an extended chain of iron-sulfur clusters, defining the structural basis for bifurcating and confurcating electron flow. These findings fundamentally revise the physiological role of HoxEFUYH by showing that photosynthetic H2 production does not rely solely on photosynthetic electrons but instead couples reduced ferredoxin from the light reaction with NADH derived from "dark" carbohydrate oxidation. This requires reassessment of current strategies for green H2 production in cyanobacteria.

biochemistry↗

bioRxiv2026

Protein Function Prediction with Pretrained ProtT5 Embeddings and Gradient Boosting

Protein function prediction remains a central challenge in computational biology due to the extreme sparsity and long-tail distribution of Gene Ontology (GO) [1] annotations. Advances in protein language models enable the extraction of dense, fixed-length representations from amino acid sequences, offering a scalable alternative to hand-picked features such as physicochemical properties. In this work, we evaluate a transformer-based embedding approach using ProtT5-XL combined with classical and modern multi-label classifiers for Gene Ontology prediction in the CAFA-6 setting. Fixed-length embeddings were generated via mean pooling of transformer hidden states and used as input to one-vs-rest logistic regression, gradient-boosted decision trees, and a neural network. Models were evaluated on held-out validation data with a focus on threshold selection, prediction sparsity, and behavior across frequent and rare GO terms. Gradient boosting consistently provided the best balance between predictive performance and stable prediction behavior, motivating its use for ontology-specific predictors across molecular function, biological process, and cellular component annotations. This study highlights practical modeling choices for large-scale protein function prediction using pretrained sequence embeddings and provides an interpretable baseline for future CAFA evaluations.

bioinformatics↗

bioRxiv2026

Comprehensive study on ferredoxin isoforms in the cyanobacterium Synechocystis sp. PCC 6803

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

biochemistry↗

bioRxiv2021

Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. Both the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex catalyze this reaction. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic conditions while the PDH complex is inactivated under the same conditions. We found that cells rely on a general shift from utilizing NAD(H)-dependent to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.

evolutionary biology↗

bioRxiv2020

Functional requirement of the Arabidopsis importin-α nuclear transport receptor family in autoimmunity mediated by the NLR protein SNC1

IMPORTIN-3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin- isoforms in Arabidopsis that recruit nuclear localization signal (NLS)-containing cargo proteins to the nuclear import machinery. IMP-3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat (NLR) immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin- genes to both types of immune responses, and we analyzed potential interactions of all importin- isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in N. benthamiana we provide evidence that among the nine -importins in Arabidopsis, IMP-3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis. SIGNIFICANCE STATEMENTSpecific requirement for the Arabidopsis -importin MOS6 in snc1-mediated autoimmunity is explained by selective formation of MOS6-SNC1 nuclear import complexes.

plant biology↗