Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe-4S] Clusters.

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

Identification and characterization of the low molecular mass ferredoxins involved in central metabolism in Heliomicrobium modesticaldum.

The homodimeric Type I reaction center (RC) from Heliomicrobium modesticaldum lacks the PsaC subunit found in Photosystem I and instead uses the interpolypeptide [4Fe-4S] cluster FX as the terminal electron acceptor. Our goal was to identify which of the small mobile dicluster ferredoxins encoded by the H. modesticaldum genome are capable of accepting electrons from the heliobacterial RC (HbRC) and pyruvate:ferredoxin oxidoreductase (PFOR), a key metabolic enzyme. Analysis of the genome revealed seven candidates: HM1_1462 (PshB1), HM1_1461 (PshB2), HM1_2505 (Fdx3), HM1_0869 (FdxB), HM1_1043, HM1_0357, and HM1_2767. Heterologous expression in Escherichia coli and studies using time-resolved optical spectroscopy revealed that only PshB1, PshB2, and Fdx3 are capable of accepting electrons from the HbRC and PFOR. Modeling studies using AlphaFold show that only PshB1, PshB2, and Fdx3 should be capable of docking on PFOR at a positively charged patch that overlays a surface-proximal [4Fe-4S] cluster. Proteomic analysis of wild-type and gene deletion strains ΔpshB1, ΔpshB2, ΔpshB1pshB2, and Δfdx3 grown under nitrogen-replete conditions revealed that Fdx3 is undetectable in the wild-type, ΔpshB1, and Δfdx3 strains, but it is present in the ΔpshB2 and ΔpshB1pshB2 strains, implying that Fdx3 may substitute for PshB2. When grown under nitrogen-deplete conditions, Fdx3 is present in the wild-type and all deletion strains except for Δfdx3. None of the knockout strains demonstrated significant impairment during chemotrophic dark growth on pyruvate, photoheterotrophic light growth on pyruvate, or phototrophic growth on acetate+CO2, indicating a high degree of redundancy among these three electron transfer proteins. Loss of both PshB1 and PshB2, but not FdxB, resulted in poor growth under N2-fixing conditions.

Acclimation of the photosynthetic apparatus to low light in a thermophilic Synechococcus sp. strain.

Depending upon their growth responses to high and low irradiance, respectively, thermophilic Synechococcus sp. isolates from microbial mats associated with the effluent channels of Mushroom Spring, an alkaline siliceous hot spring in Yellowstone National Park, can be described as either high-light (HL) or low-light (LL) ecotypes. Strains isolated from the bottom of the photic zone grow more rapidly at low irradiance compared to strains isolated from the uppermost layer of the mat, which conversely grow better at high irradiance. The LL-ecotypes develop far-red absorbance and fluorescence emission features after growth in LL. These isolates have a unique gene cluster that encodes a putative cyanobacteriochrome denoted LcyA, a putative sensor histidine kinase; an allophycocyanin (FRL-AP; ApcD4-ApcB3) that absorbs far-red light; and a putative chlorophyll a-binding protein, denoted IsiX, which is homologous to IsiA. The emergence of FRL absorbance in LL-adapted cells of Synechococcus sp. strain A1463 was analyzed in cultures responding to differences in light intensity. The far-red absorbance phenotype arises from expression of a novel antenna complex containing the FRL-AP, ApcD4-ApcB3, which is produced when cells were grown at very low irradiance. Additionally, the two GAF domains of LcyA were shown to bind phycocyanobilin and a [4Fe-4S] cluster, respectively. These ligands potentially enable this photoreceptor to respond to a variety of environmental factors including irradiance, redox potential, and/or oxygen concentration. The products of the gene clusters specific to LL-ecotypes likely facilitate growth in low-light environments through a process called Low-Light Photoacclimation.

Differential sensitivity to oxygen among the bacteriochlorophylls g in the type-I reaction centers of Heliobacterium modesticaldum.

The type-I, homodimeric photosynthetic reaction center (RC) of Heliobacteria (HbRC) is the only known RC in which bacteriochlorophyll g (BChl g) is found. It is also simpler than other RCs, having the smallest number of protein subunits and bound chromophores of any type-I RC. In the presence of oxygen, BChl g isomerizes to 81-hydroxychlorophyll aF (Chl aF). This naturally occurring process provides a way of altering the chlorophylls and studying the effect of these changes on energy and electron transfer. Transient absorbance difference spectroscopy reveals that triplet-state formation occurs in the antenna chlorophylls of HbRCs but does not provide site-specific information. Here, we report on an extended optically detected magnetic resonance (ODMR) study of the antenna triplet states in HbRCs with differing levels of conversion of BChl g to Chl aF. The data reveal pools of BChl g molecules with different triplet zero-field splitting parameters and different susceptibilities to chemical oxidation. By relating the detailed spectroscopic characteristics derived from the ODMR data to the recently solved crystallographic structure, we have tentatively identified BChl g molecules in which the probability of triplet formation is high and sites at which BChl g conversion is more likely, providing useful information about the fate of the excitation in the complex.

Designing a modified clostridial 2[4Fe-4S] ferredoxin as a redox coupler to directly link photosystem I with a Pt nanoparticle.

A methodology previously developed in our laboratory utilized an aliphatic hydrocarbon terminated by thiol groups to tether two redox proteins, i.e., the [4Fe-4S] cluster FB of photosystem I (PS I) and the distal [4Fe-4S] cluster of a [FeFe]-hydrogenase, to create a biohybrid dihydrogen-generating complex. These studies guided the design of a modified 2[4Fe-4S] cluster ferredoxin from Clostridium pasteurianum (CpFd) containing two externally facing cysteine residues in close proximity to each [4Fe-4S] cluster that replaces the aliphatic hydrocarbon dithiol tether. The advantage of using a protein is the potential to create a coupled dihydrogen-generating system in vivo. The wild-type CpFdWT and variants CpFdS11C/D40C, CpFdP20C/P49C, CpFdD7S/D36S, CpFdS11C/D40C/D7S/D36S and CpFdP20C/P49C/D7S/D36S were expressed in Escherichia coli and found to contain ~ 8 Fe and ~ 8 S atoms. The absorption spectra of the wild-type and CpFd variants displayed a peak centered at ~ 390 nm characteristic of a S → Fe charge transfer band that diminishes upon reduction with Na-dithionite. Low-temperature X-band EPR studies of the Na-dithionite-reduced wild-type and CpFd variants showed a complex spectrum indicative of two magnetically coupled [4Fe-4S]1+ clusters. EPR-monitored redox titrations of CpFdWT, CpFdD7S/D36S, CpFdS11C/D40C, CpFdP20C/P49C, CpFdS11C/D40C/D7S/D36S and CpFdP20C/P49C/D7S/D36S revealed redox potentials of - 412 ± 8 mV, - 395 ± 4 mV, - 408 ± 7 mV, - 426 ± 11 mV, - 384 ± 4 mV and - 423 ± 4 mV, respectively. The in vitro PS I-CpFdS11C/D40C/D7S/D36S-Pt nanoparticle complex was the highest performer, generating dihydrogen at a rate of 3.25 µmol H2 mg Chl-1 h-1 or 278.8 mol H2 mol PS I-1 h-1 under continuous illumination.

Structure and function of an unusual flavodoxin from the domain Archaea.

Flavodoxins, electron transfer proteins essential for diverse metabolisms in microbes from the domain Bacteria, are extensively characterized. Remarkably, although genomic annotations of flavodoxins are widespread in microbes from the domain Archaea, none have been isolated and characterized. Herein is described the structural, biochemical, and physiological characterization of an unusual flavodoxin (FldA) from Methanosarcina acetivorans, an acetate-utilizing methane-producing microbe of the domain Archaea In contrast to all flavodoxins, FldA is homodimeric, markedly less acidic, and stabilizes an anionic semiquinone. The crystal structure reveals an flavin mononucleotide (FMN) binding site unique from all other flavodoxins that provides a rationale for stabilization of the anionic semiquinone and a remarkably low reduction potentials for both the oxidized/semiquinone (-301 mV) and semiquinone/hydroquinone couples (-464 mV). FldA is up-regulated in acetate-grown versus methanol-grown cells and shown here to substitute for ferredoxin in mediating the transfer of low potential electrons from the carbonyl of acetate to the membrane-bound electron transport chain that generates ion gradients driving ATP synthesis. FldA offers potential advantages over ferredoxin by (i) sparing iron for abundant iron-sulfur proteins essential for acetotrophic growth and (ii) resilience to oxidative damage.

Expression, purification and characterization of an active C491G variant of ferredoxin sulfite reductase from Synechococcus elongatus PCC 7942.

Recently developed molecular wire technology takes advantage of [4Fe-4S] clusters that are ligated by at least one surface exposed Cys residue. Mutagenesis of this Cys residue to a Gly opens an exchangeable coordination site to a corner iron atom that can be chemically rescued by an external thiolate ligand. This ligand can be subsequently displaced by mass action using a dithiol molecular wire to tether two redox active proteins. We intend to apply this technique to tethering Photosystem I to ferredoxin sulfite reductase (FdSiR), an enzyme that catalyzes the six-electron reduction of sulfite to hydrogen sulfite and nitrite to ammonia. The enzyme contains a [4Fe-4S]2+/1+ cluster and a siroheme active site. FdSiRWT and an FdSiRC491G variant were cloned from Synechococcus elongatus PCC 7942 and expressed along with the cysG gene from Salmonella typhimurium using the pCDFDuet plasmid. UV/Vis absorbance spectra of both FdSiRWT and the FdSiRC491G variant displayed characteristic peaks at 278, 392 (Soret), 585 (α) and 714 nm (charge transfer band), and 278, 394 (Soret), 587 (α) and 714 nm (charge transfer band) respectively. Both enzymes in their as-isolated forms displayed an EPR spectrum characteristic of an S = 5/2 high spin heme. When reduced, both enzymes exhibited the signal of a low spin S = 1/2 [4Fe-4S]1+ cluster. The FdSiRWT and FdSiRC491G variant both showed activity using reduced methyl viologen and Synechococcus elongatus PCC 7942 ferredoxin 1 (Fd1) as electron donors. Based on these results, the FdSIRC491G variant should be a suitable candidate for wiring to Photosystem I.

Toward a mechanistic and physiological understanding of a ferredoxin:disulfide reductase from the domains Archaea and Bacteria.

Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster. UV-visible, EPR, and Mössbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]1+ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR, advancing the physiological understanding of FDR's role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show that FDR homologs are widespread in diverse microbes from the domain Bacteria.

Presence of a [3Fe-4S] cluster in a PsaC variant as a functional component of the photosystem I electron transfer chain in Synechococcus sp. PCC 7002.

A site-directed C14G mutation was introduced into the stromal PsaC subunit of Synechococcus sp. strain PCC 7002 in vivo in order to introduce an exchangeable coordination site into the terminal FB [4Fe-4S] cluster of Photosystem I (PSI). Using an engineered PSI-less strain (psaAB deletion), psaC was deleted and replaced with recombinant versions controlled by a strong promoter, and the psaAB deletion was complemented. Modified PSI accumulated at lower levels in this strain and supported slower photoautotrophic growth than wild type. As-isolated PSI complexes containing PsaCC14G showed resonances with g values of 2.038 and 2.007 characteristic of a [3Fe-4S]1+ cluster. When the PSI complexes were illuminated at 15 K, these resonances partially disappeared and two new sets of resonances appeared. The majority set had g values of 2.05, 1.95, and 1.85, characteristic of FA-, and the minority set had g values of 2.11, 1.90, and 1.88 from FB' in the modified site. The S = 1/2 spin state of the latter implied the presence of a thiolate as the terminal ligand. The [3Fe-4S] clusters could be partially reconstituted with iron, producing a larger population of [4Fe-4S] clusters. Rates of flavodoxin reduction were identical in PSI complexes isolated from wild type and the PsaCC14G variant strain; this implied equivalent capacity for forward electron transfer in PSI complexes that contained [3Fe-4S] and [4Fe-4S] clusters. The development of this cyanobacterial strain is a first step toward translation of in vitro PSI-based biosolar molecular wire systems in vivo and provides new insights into the formation of Fe/S clusters.

Thermodynamics of the Electron Acceptors in Heliobacterium modesticaldum: An Exemplar of an Early Homodimeric Type I Photosynthetic Reaction Center.

The homodimeric type I reaction center in heliobacteria is arguably the simplest known pigment-protein complex capable of conducting (bacterio)chlorophyll-based conversion of light into chemical energy. Despite its structural simplicity, the thermodynamics of the electron transfer cofactors on the acceptor side have not been fully investigated. In this work, we measured the midpoint potential of the terminal [4Fe-4S](2+/1+) cluster (FX) in reaction centers from Heliobacterium modesticaldum. The FX cluster was titrated chemically and monitored by (i) the decrease in the level of stable P800 photobleaching by optical spectroscopy, (ii) the loss of the light-induced g ≈ 2 radical from P800(+•) following a single-turnover flash, (iii) the increase in the low-field resonance at 140 mT attributed to the S = (3)/2 ground spin state of FX(-), and (iv) the loss of the spin-correlated P800(+) FX(-) radical pair following a single-turnover flash. These four techniques led to similar estimations of the midpoint potential for FX of -502 ± 3 mV (n = 0.99), -496 ± 2 mV (n = 0.99), -517 ± 10 mV (n = 0.65), and -501 ± 4 mV (n = 0.84), respectively, with a consensus value of -504 ± 10 mV (converging to n = 1). Under conditions in which FX is reduced, the long-lived (∼15 ms) P800(+) FX(-) state is replaced by a rapidly recombining (∼15 ns) P800(+)A0(-) state, as shown by ultrafast optical experiments. There was no evidence of the presence of a P800(+) A1(-) spin-correlated radical pair by electron paramagnetic resonance (EPR) under these conditions. The midpoint potentials of the two [4Fe-4S](2+/1+) clusters in the low-molecular mass ferredoxins were found to be -480 ± 11 mV/-524 ± 13 mV for PshBI, -453 ± 6 mV/-527 ± 6 mV for PshBII, and -452 ± 5 mV/-533 ± 8 mV for HM1_2505 as determined by EPR spectroscopy. FX is therefore suitably poised to reduce one [4Fe-4S](2+/1+) cluster in these mobile electron carriers. Using the measured midpoint potential of FX and a quasi-equilibrium model of charge recombination, the midpoint potential of A0 was estimated to be -854 mV at room temperature. The midpoint potentials of A0 and FX are therefore 150-200 mV less reducing than their respective counterparts in Photosystem I of cyanobacteria and plants. This places the redox potential of the FX cluster in heliobacteria approximately equipotential to the highest-potential iron-sulfur cluster (FA) in Photosystem I, consistent with its assignment as the terminal electron acceptor.

Tetratricopeptide repeat protein protects photosystem I from oxidative disruption during assembly.

A Chlamydomonas reinhardtii mutant lacking CGL71, a thylakoid membrane protein previously shown to be involved in photosystem I (PSI) accumulation, exhibited photosensitivity and highly reduced abundance of PSI under photoheterotrophic conditions. Remarkably, the PSI content of this mutant declined to nearly undetectable levels under dark, oxic conditions, demonstrating that reduced PSI accumulation in the mutant is not strictly the result of photodamage. Furthermore, PSI returns to nearly wild-type levels when the O2 concentration in the medium is lowered. Overall, our results suggest that the accumulation of PSI in the mutant correlates with the redox state of the stroma rather than photodamage and that CGL71 functions under atmospheric O2 conditions to allow stable assembly of PSI. These findings may reflect the history of the Earth's atmosphere as it transitioned from anoxic to highly oxic (1-2 billion years ago), a change that required organisms to evolve mechanisms to assist in the assembly and stability of proteins or complexes with O2-sensitive cofactors.

Spectroscopic characterization of the SH2- and active site-directed peptide sequences of a bivalent Src kinase inhibitor.

The spectral properties of the SH2 and active site-directed sequences of the bivalent Src kinase inhibitor Ac-EELL(F5)Phe-(GABA)3-pYEEIE-amide (1) have been determined. Ac-pYEEIE-amide (2) and AcEELL(F5)Phe-amide (3), as well as the amino acids phosphotyrosine (pTyr) and pentafluorophenylalanine (F5)Phe, have been characterized by electronic absorption, fluorescence, and vibrational spectroscopy. Specific and unique marker bands that originate from the phosphate group of pTyr and the fluorinated aromatic ring of (F5)Phe have been identified, with the latter showing some solvent dependence. Peptide 2 was found to have excitation and emission wavelengths emanating from pTyr at 268 and 295 nm, respectively, whereas peptide 3 displayed excitation and emission peaks attributable to (F5)Phe at 274 and 315 nm, respectively. Fourier transform infrared (FT-IR) analysis of the amino acid pTyr identified distinct marker bands at approximately 930, 1090, and 1330 cm(-1) that could be attributed to the phosphate group. These markers were also observed in the IR spectrum of peptide 2. Likewise, peptide 3 displayed a characteristic C-F stretching mode at 961 cm(-1) due to the presence of (F5)Phe, including two C-F reporting ring modes at 1509 and 1527 cm(-1). Identifying and monitoring spectroscopic changes in these marker bands may afford a means to observe the molecular interactions that occur when peptides 1-3 bind to the Src kinase.