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PreprintCited by 6Photosynthetic Processes and MechanismsMetalloenzymes and iron-sulfur proteinsMetal-Catalyzed Oxygenation Mechanisms

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Physcomitrium patens flavodiiron proteins form a redox-dependent heterocomplex

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Beraldo, Claudia, Traverso, Eleonora, Boschin, Marco, Cendron, Laura, Morosinotto, Tomas, Alboresi, Alessandro·2024·Journal of Biological Chemistry
DOI 10.1101/2024.01.29.577648PMC PMC11408035

Preprint — published as 10.1016/j.jbc.2024.107643

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Abstract

Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by exploiting electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens . The two proteins, expressed and purified from Escherichia coli , bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero multimer protein complex, that has never been isolated and characterized so far. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo role and regulation.

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Figure 1 Predicted structure analysis of Physcomitrium patens FLVA and FLVB.A, schematic representation and AlphaFold model of FLVA (A9RYP3) and (B) FLVB (A9RQ00) structures. Dotted lines indicate the predicted cleavage sites for FLVA (position 70) and FLVB (position 65) chloroplast target peptides. The N-terminal metallo-β-lactamase domain contains a diiron center (Fe-Fe), the flavodoxin-like domain harbors a flavin mononucleotide (FMN), and the C-terminal domain is a flavin reductase-like domain (FlvR). The three domains are shown respectively in orange, yellow, and green. FLVA-specific residues (I313-K364) are shown in blue. Iron atoms (Fe) are represented as red spheres present in the active site.
Figure 1 Predicted structure analysis of Physcomitrium patens FLVA and FLVB.A, schematic representation and AlphaFold model of FLVA (A9RYP3) and (B) FLVB (A9RQ00) structures. Dotted lines indicate the predicted cleavage sites for FLVA (position 70) and FLVB (position 65) chloroplast target peptides. The N-terminal metallo-β-lactamase domain contains a diiron center (Fe-Fe), the flavodoxin-like domain harbors a flavin mononucleotide (FMN), and the C-terminal domain is a flavin reductase-like domain (FlvR). The three domains are shown respectively in orange, yellow, and green. FLVA-specific residues (I313-K364) are shown in blue. Iron atoms (Fe) are represented as red spheres present in the active site.CC BY-NC-ND · PubMed Central
Figure 2 FLVA and FLVB biochemical properties. FLVA and FLVB biochemical properties. Coomassie Brilliant Blue-stained SDS-PAGE (CBB) of purified FLVA (A) and FLVB (B) and immunoblot analysis (IB) performed with antisera anti-FLVA and anti-Strep-Tag (A) and anti-FLVB and anti-His-Tag. On the left side, the apparent molecular weight of the ladder is reported in kDa. UV-visible absorption spectra of purified FLVA (C) and FLVB (D). Absorption spectra of approximately 5 μM protein in a solution containing 30 mM Tris and 300 mM NaCl at pH 8.0 were recorded at room temperature.
Figure 2 FLVA and FLVB biochemical properties. FLVA and FLVB biochemical properties. Coomassie Brilliant Blue-stained SDS-PAGE (CBB) of purified FLVA (A) and FLVB (B) and immunoblot analysis (IB) performed with antisera anti-FLVA and anti-Strep-Tag (A) and anti-FLVB and anti-His-Tag. On the left side, the apparent molecular weight of the ladder is reported in kDa. UV-visible absorption spectra of purified FLVA (C) and FLVB (D). Absorption spectra of approximately 5 μM protein in a solution containing 30 mM Tris and 300 mM NaCl at pH 8.0 were recorded at room temperature.CC BY-NC-ND · PubMed Central
Figure 3 Kinetics of FLVA and FLVB NAD(P)H oxidase activity. FLVA and FLVB activity were tested in the presence of either 150 μM NADH (A) or NADPH (B). Red lines correspond to 7 μM FLVA and blue lines correspond to 7 μM FLVB. Black lines correspond to the negative control in the absence of FLV proteins. Traces were shifted to OD340nm equal to 1 as a starting point immediately after FLV protein addition. Absorption spectra were recorded at room temperature. C-D, Hill fitting kinetics and fitted values for purified FLVA and FLVB in the presence of NADH. The solid line indicates Hill fitting curve of FLVA (C) or FLVB (D). n = 3 reactions were performed in the presence of FLVA and different concentrations of NADH, n = 4 reactions were performed in the presence of FLVB and different concentrations of NADH. Mean values are represented ± SEM.
Figure 3 Kinetics of FLVA and FLVB NAD(P)H oxidase activity. FLVA and FLVB activity were tested in the presence of either 150 μM NADH (A) or NADPH (B). Red lines correspond to 7 μM FLVA and blue lines correspond to 7 μM FLVB. Black lines correspond to the negative control in the absence of FLV proteins. Traces were shifted to OD340nm equal to 1 as a starting point immediately after FLV protein addition. Absorption spectra were recorded at room temperature. C-D, Hill fitting kinetics and fitted values for purified FLVA and FLVB in the presence of NADH. The solid line indicates Hill fitting curve of FLVA (C) or FLVB (D). n = 3 reactions were performed in the presence of FLVA and different concentrations of NADH, n = 4 reactions were performed in the presence of FLVB and different concentrations of NADH. Mean values are represented ± SEM.CC BY-NC-ND · PubMed Central
Figure 4 High-resolution respirometry of P. patens FLVB performed in an Oroboros Oxygraph-2k. The measurement started with sample buffer (Tris 30 mM and NaCl 300 mM) either containing 6 μM FLVB (solid line) or not (dashed line). After about 7.5 min 150 μM NADH was added to both samples. To verify protein stability, 70 μM NADH was added again after about 35 min.
Figure 4 High-resolution respirometry of P. patens FLVB performed in an Oroboros Oxygraph-2k. The measurement started with sample buffer (Tris 30 mM and NaCl 300 mM) either containing 6 μM FLVB (solid line) or not (dashed line). After about 7.5 min 150 μM NADH was added to both samples. To verify protein stability, 70 μM NADH was added again after about 35 min.CC BY-NC-ND · PubMed Central
Figure 5 FLVA/FLVB heterocomplex biochemical and functional properties.A, representative Coomassie Brilliant Blue-stained SDS-polyacrylamide gel and Western blot analysis of purified heterocomplex using α-FLVA and α-FLVB, α-StrepTag, α-HisTag antibodies. On the left side, the apparent molecular weight of the ladder is reported in kDa. B, the absorption spectrum of approximately 5 μM FLVA/B in a solution containing 30 mM Tris and 300 mM NaCl at pH 8.0 was recorded at room temperature. C, NAD(P)H oxidase activity of 1 μM FLVA/B heterocomplex in the presence of 150 μM NADH (green line) and 150 μM NADPH (orange line). Traces were shifted to OD340nm equal to 1 as the starting point immediately after protein adding. Absorption spectra were recorded at room temperature. D, Hill fitting Kinetics and fitted values for purified FLVA/B in the presence of NADH. The solid line indicates the Hill fitting curve. n = 3 Values are expressed ± SEM.
Figure 5 FLVA/FLVB heterocomplex biochemical and functional properties.A, representative Coomassie Brilliant Blue-stained SDS-polyacrylamide gel and Western blot analysis of purified heterocomplex using α-FLVA and α-FLVB, α-StrepTag, α-HisTag antibodies. On the left side, the apparent molecular weight of the ladder is reported in kDa. B, the absorption spectrum of approximately 5 μM FLVA/B in a solution containing 30 mM Tris and 300 mM NaCl at pH 8.0 was recorded at room temperature. C, NAD(P)H oxidase activity of 1 μM FLVA/B heterocomplex in the presence of 150 μM NADH (green line) and 150 μM NADPH (orange line). Traces were shifted to OD340nm equal to 1 as the starting point immediately after protein adding. Absorption spectra were recorded at room temperature. D, Hill fitting Kinetics and fitted values for purified FLVA/B in the presence of NADH. The solid line indicates the Hill fitting curve. n = 3 Values are expressed ± SEM.CC BY-NC-ND · PubMed Central
Figure 6 Redox dependence of flavodiiron proteins heterocomplex.A, SDS-PAGE and Western blotting of purified FLVA/B heterocomplex in the presence or absence of reducing agent. On the left side, the apparent molecular weight of the ladder is reported in kDa. The samples were incubated at room temperature in the presence or absence of DTT as indicated on the top of each lane. Prior to SDS-PAGE all samples were heated at 100 °C. B, FLVA/B heterocomplex NADH oxidase activity in the presence (red line) or absence (green line) of reducing agent. 4 μM of purified enzyme and the equivalent volume of protein buffer (black line) were incubated at room temperature for ∼1 h with DTT prior to activity test. 150 μM NADH without adding FLV and DTT was also monitored as a control (black line). Traces were shifted to OD340nm equal to 1 as the starting point immediately after protein adding. Absorption spectra were recorded at room temperature.
Figure 6 Redox dependence of flavodiiron proteins heterocomplex.A, SDS-PAGE and Western blotting of purified FLVA/B heterocomplex in the presence or absence of reducing agent. On the left side, the apparent molecular weight of the ladder is reported in kDa. The samples were incubated at room temperature in the presence or absence of DTT as indicated on the top of each lane. Prior to SDS-PAGE all samples were heated at 100 °C. B, FLVA/B heterocomplex NADH oxidase activity in the presence (red line) or absence (green line) of reducing agent. 4 μM of purified enzyme and the equivalent volume of protein buffer (black line) were incubated at room temperature for ∼1 h with DTT prior to activity test. 150 μM NADH without adding FLV and DTT was also monitored as a control (black line). Traces were shifted to OD340nm equal to 1 as the starting point immediately after protein adding. Absorption spectra were recorded at room temperature.CC BY-NC-ND · PubMed Central
Figure 7 Proposed model describing P. patens flavodiiron protein catalytic activity.A, overall proposed reaction scheme. Four electrons are necessary for the complete reduction of O2 to water, two electrons reduce the FMN moiety, while the other two the Fe-Fe center. B, FLVA and FLVB form stable bound heterotetramer in the stroma of P. patens chloroplasts. The dashed line indicates electron flow. Electrons coming from Fd and NADPH pool could participate in FLV oxygen reduction reaction.
Figure 7 Proposed model describing P. patens flavodiiron protein catalytic activity.A, overall proposed reaction scheme. Four electrons are necessary for the complete reduction of O2 to water, two electrons reduce the FMN moiety, while the other two the Fe-Fe center. B, FLVA and FLVB form stable bound heterotetramer in the stroma of P. patens chloroplasts. The dashed line indicates electron flow. Electrons coming from Fd and NADPH pool could participate in FLV oxygen reduction reaction.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Beraldo, C., Traverso, E., Boschin, M., Cendron, L., Morosinotto, T., & Alboresi, A.
Affiliations
University of Padua (IT)
Corresponding author
Alboresi, Alessandro
ORCID
Venue
Journal of Biological Chemistry
Publisher
openRxiv
Published
2024-01-29
Volume
300
Issue
9
Pages
107643-107643
ISSN
0021-9258 (ISSN-L)
DOI
10.1101/2024.01.29.577648
PMC
PMC11408035
OpenAlex
W4401438711
Cited by
6
References
1
Citations by year
2026: 1 · 2025: 5
FWCI
0.82
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by)
Funders
  • European Commission (H2020, CN00000033, 859770)
  • Ministero dell’Istruzione, dell’Università e della Ricerca
  • Horizon 2020 Framework Programme (859770)
  • Ministero dell'Istruzione e del Merito
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Metalloenzymes and iron-sulfur proteins
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
Keywords
flavodiiron proteins, Physcomitrella patens, photosystem I, oxygen reduction reaction, protein oligomerization, tandem affinity purification, photosynthetic electron transport
MeSH terms
Escherichia coli, Iron, Plant Proteins, Bryopsida, Photosystem I Protein Complex, Protein Multimerization

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CoChat. (2026). Citation check: Physcomitrium patens flavodiiron proteins form a redox-dependent heterocomplex. https://app.cochat.ai/papers/10.1101/2024.01.29.577648
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Beraldo, C., Traverso, E., Boschin, M., Cendron, L., Morosinotto, T., & Alboresi, A. (2024). Physcomitrium patens flavodiiron proteins form a redox-dependent heterocomplex. Journal of Biological Chemistry. https://doi.org/10.1101/2024.01.29.577648