Recombination of 2Fe-2S Ferredoxins Reveals Differences in the Inheritance of Thermostability and Midpoint Potential

A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.

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In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

Applied and Environmental Microbiology ◽

10.1128/aem.71.3.1522-1530.2005 ◽

2005 ◽

Vol 71 (3) ◽

pp. 1522-1530 ◽

Cited By ~ 40

Author(s):

Amy M. Grunden ◽

Francis E. Jenney ◽

Kesen Ma ◽

Mikyoung Ji ◽

Michael V. Weinberg ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Pyrococcus Furiosus ◽

Expression System ◽

Anaerobic Bacteria ◽

Flavin Mononucleotide ◽

Superoxide Reductase ◽

Recombinant Enzymes ◽

Significant Similarity ◽

Midpoint Potential

ABSTRACT A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of −173 mV at 23°C (−193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.

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Reduction of the oxidized bacteriochlorophyll dimer in reaction centers by ferrocene is dependent upon the driving force

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424607000266 ◽

2007 ◽

Vol 11 (03) ◽

pp. 205-211 ◽

Cited By ~ 3

Author(s):

László Kálmán ◽

Arlene L. M. Haffa ◽

JoAnn C. Williams ◽

Neal W. Woodbury ◽

James P. Allen

Keyword(s):

Electron Transfer ◽

Rate Constants ◽

Ph Dependence ◽

Photosynthetic Bacterium ◽

Energy Difference ◽

Reaction Centers ◽

Free Energy Difference ◽

Midpoint Potential ◽

Bacteriochlorophyll Dimer ◽

Purple Photosynthetic Bacterium

The rates of electron transfer from ferrocene to the oxidized bacteriochlorophyll dimer, P , in reaction centers from the purple photosynthetic bacterium Rhodobacter sphaeroides, were measured for a series of mutants in which the P / P + midpoint potentials range from 410 to 765 mV (Lin et al. Proc. Natl. Acad. Sci. USA 1994; 91: 10265-10269). The observed rate constant for each mutant was found to be linearly dependent upon the ferrocene concentration up to 50 μM. The electron transfer is described as a second order reaction with rate constants increasing from 1.5 to 35 × 106 M -1. s -1 with increasing P / P + midpoint potential. This dependence was tested for three additional mutants, each of which exhibits a pH dependence of the P / P + midpoint potential due to an electrostatic interaction with an introduced carboxylic group (Williams et al. Biochemistry 2001; 40: 15403-15407). For these mutants, the pH dependence of the bimolecular rate constants followed a sigmoidal pattern that could be described with a Henderson-Hasselbalch equation, attributable to the change of the free energy difference for the reaction due to deprotonation of the introduced carboxylic side chains.

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Cytochrome c550 from Pseudomonas aeruginosa

Biochemical Journal ◽

10.1042/bj2890173 ◽

1993 ◽

Vol 289 (1) ◽

pp. 173-178 ◽

Cited By ~ 16

Author(s):

P Reichmann ◽

H Görisch

Keyword(s):

Pseudomonas Aeruginosa ◽

Electron Transfer ◽

Oxygen Consumption ◽

Electron Transport ◽

Electron Transport Chain ◽

Prosthetic Group ◽

Midpoint Potential ◽

Transport Chain ◽

Ethanol Dehydrogenase ◽

Soluble C

In cells of Pseudomonas aeruginosa A.T.C.C. 17933 grown on ethanol the synthesis of a soluble c-type cytochrome, together with quinoprotein ethanol dehydrogenase, is induced. The cytochrome, with an alpha-absorption band at 550 nm, was purified to homogeneity. The molecular mass of the monomeric protein is 15 kDa, the pI is 4.8, and it contains one haem prosthetic group. The midpoint potential of the autoxidizable, but not autoreducible, cytochrome is 280 mV. Cytochrome c550 mediates electron transfer between quinoprotein ethanol dehydrogenase and ferricyanide. In a system composed of membrane particles with NN‘NN’-tetramethyl-p-phenylenediamine oxidase activity and quinoprotein ethanol dehydrogenase, oxygen consumption is only observed in the presence of cytochrome c550. This indicates the participation of the cytochrome in the electron-transport chain linked to quinoprotein ethanol dehydrogenase in P. aeruginosa. The electron transport from ethanol dehydrogenase to oxygen is inhibited by myxothiazol and antimycin, indicating that a cytochrome bc1-like complex is involved.

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A thylakoid membrane-bound and redox-active rubredoxin (RBD1) functions in de novo assembly and repair of photosystem II

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903314116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16631-16640 ◽

Cited By ~ 4

Author(s):

José G. García-Cerdán ◽

Ariel L. Furst ◽

Kent L. McDonald ◽

Danja Schünemann ◽

Matthew B. Francis ◽

...

Keyword(s):

Photosystem Ii ◽

Thylakoid Membrane ◽

De Novo Assembly ◽

Biochemical Characterization ◽

De Novo ◽

Midpoint Potential ◽

Photosynthetic Organisms ◽

Redox Active ◽

Photooxidative Damage

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559. Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.

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Electrochemical, Spectroscopic, and Computational Investigations on Redox Reactions of Selenium Species on Galena Surfaces

Minerals ◽

10.3390/min9070437 ◽

2019 ◽

Vol 9 (7) ◽

pp. 437 ◽

Cited By ~ 1

Author(s):

Peter Cook ◽

YoungJae Kim ◽

Ke Yuan ◽

Maria C. Marcano ◽

Udo Becker

Keyword(s):

Photoelectron Spectroscopy ◽

Redox Reactions ◽

Redox Potentials ◽

Free Energies ◽

Cathodic Peak ◽

Redox Transformation ◽

Hydrogen Electrode ◽

Midpoint Potential ◽

Ph Conditions ◽

Cv Measurements

Despite previous studies investigating selenium (Se) redox reactions in the presence of semiconducting minerals, Se redox reactions mediated by galena (PbS) are poorly understood. In this study, the redox chemistry of Se on galena is investigated over a range of environmentally relevant Eh and pH conditions (+0.3 to −0.6 V vs. standard hydrogen electrode, SHE; pH 4.6) using a combination of electrochemical, spectroscopic, and computational approaches. Cyclic voltammetry (CV) measurements reveal one anodic/cathodic peak pair at a midpoint potential of +30 mV (vs. SHE) that represents reduction and oxidation between HSeO3− and H2Se/HSe−. Two peak pairs with midpoint potentials of −400 and −520 mV represent the redox transformation from Se(0) to HSe− and H2Se species, respectively. The changes in Gibbs free energies of adsorption of Se species on galena surfaces as a function of Se oxidation state were modeled using quantum-mechanical calculations and the resulting electrochemical peak shifts are (−0.17 eV for HSeO3−/H2Se, −0.07 eV for HSeO3−/HSe−, 0.15 eV for Se(0)/HSe−, and −0.15 eV for Se(0)/H2Se). These shifts explain deviation between Nernstian equilibrium redox potentials and observed midpoint potentials. X-ray photoelectron spectroscopy (XPS) analysis reveals the formation of Se(0) potentials below −100 mV and Se(0) and Se(−II) species at potentials below −400 mV.

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A change in the midpoint potential of the quinone QA in Photosystem II associated with photoactivation of oxygen evolution

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/0005-2728(95)00003-2 ◽

1995 ◽

Vol 1229 (2) ◽

pp. 202-207 ◽

Cited By ~ 117

Author(s):

Giles N. Johnson ◽

A.William Rutherford ◽

Anja Krieger

Keyword(s):

Photosystem Ii ◽

Oxygen Evolution ◽

Midpoint Potential

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Electron Transfer and Binding of thec-Type Cytochrome TorC to the TrimethylamineN-Oxide Reductase inEscherichia coli

Journal of Biological Chemistry ◽

10.1074/jbc.m008875200 ◽

2000 ◽

Vol 276 (15) ◽

pp. 11545-11551 ◽

Cited By ~ 69

Author(s):

Stéphanie Gon ◽

Marie-Thérèse Giudici-Orticoni ◽

Vincent Méjean ◽

Chantal Iobbi-Nivol

Keyword(s):

Electron Transfer ◽

Biochemical Characterization ◽

Reduced Form ◽

Visible Absorption ◽

Midpoint Potential ◽

Redox Potentiometry ◽

Family Based ◽

Transfer Chain

Reduction of trimethylamineN-oxide (E′0(TMAO/TMA)= +130 mV) inEscherichia coliis carried out by the Tor system, an electron transfer chain encoded by thetorCADoperon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemicc-type cytochrome TorC. Although the role of TorA in the reduction of trimethylamineN-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapCc-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorCN) to the cytochromes of this family, but TorC contains a C-terminal extension (TorCC) with an additional heme-binding site. In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO. The intact TorC protein and its two domains, TorCNand TorCC, were produced independently and purified for a biochemical characterization. The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (−177 and −98 mV) localized in the tetrahemic TorCNand one positive midpoint potential (+120 mV) in the monohemic TorCC. In agreement with these values, thein vitroreconstitution of electron transfer between TorC, TorCN, or TorCCand TorA showed that only TorC and TorCCwere capable of electron transfer to TorA. Surprisingly, interaction studies revealed that only TorC and TorCNstrongly bind TorA. Therefore, TorCCdirectly transfers electrons to TorA, whereas TorCN, which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorCCand the binding to TorA.

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