Quinol-cytochrome c oxidoreductase and cytochrome c4 mediate electron transfer during selenate respiration in Thauera selenatis

Abstract

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (E(m) + 234 +/- 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified ( approximately 24 and approximately 6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c(4) family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 +/- 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c(4) is a novel route for a member of the DMSO reductase family of molybdoenzymes.

FIGURE 1.

Subunit organization and redox cofactors of periplasmic type II molybdoenzymes. Direction of electron transfer is indicated by the arrows. Reductases include selenate and chlorate reductase. Dehydrogenases include dimethylsulfide and ethylbenzene dehydrogenases.

FIGURE 2.

Cytochrome c profile from T. selenatis. A, SDS-PAGE gels stained for c-type cytochromes, showing periplasmic cytochromes (cytc-Ts1–7) expressed during selenate and/or nitrate respiration. Lane 1, Invitrogen SeeBlue Plus2 Prestained Standard; lane 2, periplasmic fraction from selenate grown cells (heme stained); and lane 3, periplasmic fraction from nitrate grown cells (heme stained). 7 μg of protein was loaded in each lane. Lane 4, purified cytc-Ts4 (∼24 kDa) stained with Invitrogen SeeBlue Plus2; lane 5, purified cytc-Ts4 (∼24 kDa) stained with heme stain; lane 6, purified cytc-Ts7 (∼6 kDa) stained with Invitrogen SeeBlue Plus2; and lane 7, purified cytc-Ts7 (∼6 kDa) stained with heme stain. B, spectra of 25 μm cytc-Ts4 (∼24-kDa protein) as purified (solid line), reduced with sodium dithionite (dotted line), and oxidized with potassium ferricyanide (dashed line). C, spectra of 25 μm cytc-Ts7 (∼6-kDa protein) as purified (solid line), reduced with sodium dithionite (dotted line), and oxidized with potassium ferricyanide (dashed line).

FIGURE 3.

Spectroscopic assay of electron donation from cytc-Ts4 to SerABC. cytc-Ts4 (5 μm) reduced with dithionite (dashed line) was mixed with 1 μm SerABC and 20 mm selenate and re-oxidation was monitored by wavelength scanning UV-visible spectroscopy (solid line).

FIGURE 4.

Optical redox titration of cytc-Ts4. A, α and β region of cytc-Ts4 spectrum during titrations. The black arrow indicates increasing redox potential. B, fraction of cytochrome reduced as calculated with reference to absorbance at 551 nm, as a function of redox potential. Nernst curves for n = 1 electrons (dashed line) and n = 2 electrons (solid line) are also shown.

FIGURE 5.

Optical redox titration of SerC. A, the α and β region of b-heme spectrum during titrations. The black arrow indicates increasing redox potential. Samples were prepared in 30 mm Tris-HCl, pH 7.5. B, fraction of cytochrome b reduced as calculated with reference to absorbance at 558 nm, as a function of redox potential. Nernst curve for n = 1 electron.

FIGURE 6.

Inhibition of respiration by myxothiazol, antimycin A, and HQNO. A, optical density of T. selenatis cultures grown with 10 mm selenate as electron acceptor in the absence and presence of inhibitors. Shown are no inhibitor (■), 10 μm myxothiazol (♦), 20 μm HQNO (●), and both 10 μm myxothiazol and 20 μm HQNO (▾). Positive control: T. selenatis grown with 5 mm nitrite as electron acceptor in the presence of 10 μm myxothiazol (○). B, optical density of T. selenatis cultures grown with 10 mm selenate as electron acceptor in the absence and presence of antimycin A. Shown are no inhibitor (■), 10 μm antimycin A (♦), and 20 μm antimycin A (●). C, specific growth rate (μ_max) of T. selenatis cultures grown on increasing selenate concentration in the absence (■) and presence (●) of myxothiazol (10 μm). In all cases error bars represent standard deviation of n = 10 cultures.

FIGURE 7.

Schematic diagram showing the electron transport chain of T. selenatis during anaerobic growth on selenate.

FIGURE 8.

b-Heme coordination in SerC. A, predicted structure of SerC modeled upon the crystal structure of EBDH γ-subunit. The superposition shows SerC in green and the EbdC in cyan. B, detail of heme pocket showing ligands. Homology modeling was performed using the homology tools in the software package MOE^TM. The sequence of T. selenatis SerC was aligned and modeled against the structure of EBDH γ-subunit from A. aromaticum (PDB ID 2IVFC). The sequence was modeled from residues 27–239. The modeling was performed using the protein template alone, and additionally using the environment of the template structure with the heme ligand. An ensemble of 30 intermediate models was created, and the best intermediate model as minimized using the CHARM22 force field to a root mean square gradient of 0.01. The quality of the models was assessed by PROCHECK (52, 53). Analysis of the model structure and generation of molecular images were carried out using PyMOL (54).