Mutation of the heme axial ligand of Escherichia coli succinate-quinone reductase: implications for heme ligation in mitochondrial complex II from yeast

Abstract

A b-type heme is conserved in membrane-bound complex II enzymes (SQR, succinate-ubiquinone reductase). The axial ligands for the low spin heme b in Escherichia coli complex II are SdhC His84 and SdhD His71. E. coli SdhD His71 is separated by 10 residues from SdhD Asp82 and Tyr83 which are essential for ubiquinone catalysis. The same His-10x-AspTyr motif dominates in homologous SdhD proteins, except for Saccharomyces cerevisiae where a tyrosine is at the axial position (Tyr-Cys-9x-AspTyr). Nevertheless, the yeast enzyme was suggested to contain a stoichiometric amount of heme, however, with the Cys ligand in the aforementioned motif acting as heme ligand. In this report, the role of Cys residues for heme coordination in the complex II family of enzymes is addressed. Cys was substituted to the SdhD-71 position and the yeast Tyr71Cys72 motif was also recreated. The Cys71 variant retained heme, although it was high spin, while the Tyr71Cys72 mutant lacked heme. Previously the presence of heme in S. cerevisiae was detected by a spectral peak in fumarate-oxidized, dithionite-reduced mitochondria. Here it is shown that this method must be used with caution. Comparison of bovine and yeast mitochondrial membranes shows that fumarate induced reoxidation of cytochromes in both SQR and the bc1 complex (ubiquinol-cytochrome c reductase). Thus, this report raises a concern about the presence of low spin heme b in S. cerevisiae complex II.

Figure 1

Crystal structure of helix II of the E. coli SdhD subunit with heme b and ubiquinone (Protein data bank code 1NEK). A. Side chains of the residues that form a conservative motif His-10x-AspTyr and their interaction with heme b (magenta) and ubiquinone (yellow) are shown. Computer simulation: B. Top view at the heme b with His 71 substituted to a cysteine residue. C. Simulation of the double mutant: His71 substituted with Tyr and Ala72 with Cys. The figure was prepared using PyMOL (DeLano Scientific LLC, Palo Alto, CA). The mutated side chain rotamers were chosen manually based on the best alignment with original amino acid positions.

Figure 2

Comparison of the amino acid sequence of the SdhD homologous subunits from various species. Conservative positions for the His-10x-AspTyr are highlighted in bold and the His axial ligand or comparable position in yeast is underlined, as is the Cys residue in yeast Sdh4p that was proposed to ligand the heme [19].

Figure 3

Absorption difference (dithionite-reduced minus oxidized) spectra of E. coli DW35 membranes harboring SQR mutant protein. The spectra were determined at 25°C in a 1 ml cuvette containing 1 mg/ml membrane protein in 50 mM potassium phosphate (pH 7.0), 0.1 mM EDTA. Solid sodium dithionite was added to reduce the membrane suspensions. Trace a – wild type SQR; trace b- SdhD H71C; trace c – SdhD H71Y; trace d – SdhD Y71/C72. Insert shows the magnification of the α region of the same spectra.

Figure 4

Effect of carbon monoxide on the absorption difference spectra of isolated E. coli membranes. The spectra were determined as described in the legend for Fig 3. DW35 control membranes (trace a) and membranes with the SdhD H71C mutant (trace b) were reduced with dithionite and the spectra recorded. The suspensions in the cuvette were then treated with CO gas for 1 min and the spectra recorded. The CO-reduced minus dithionite-reduced difference spectra are shown. Trace c - Double difference spectrum represents subtraction of background bd-oxidase from DW35 control membranes (trace b minus trace a) to show the CO induced spectrum of the H71C mutant.

Figure 5

Reoxidation of dithionite-reduced isolated E. coli SQR by fumarate. SQR 2.4 μM was reduced by solid sodium dithionite in 1 ml of 50 mM of potassium phosphate (pH 6.5) with 30 μM UQ₁ present and subsequent addition of 20 μl of 1 M fumarate. 2 μM atpenin A5 was added before the reduction with dithionite. Absorbance changes were recorded at 558 nm.

Figure 6

Reoxidation of the dithionite reduced bovine submitochondrial particles by fumarate. SMP (0.8 mg protein/ml) were suspended in 1 ml of 0.25 M sucrose, 10 mM potassium phosphate (pH 6.5), 2 mM potassium cyanide, and 0.1% of dodecyl maltoside. Routinely 25 μM K₃Fe(CN)₆ was added to ensure complete oxidation of the cytochromes. Dashed line shows a spectrum of completely reduced membrane 2 minutes after a few grains of solid sodium dithionite was added. 20 ul of 1 M fumarate was added to the cuvette and the spectra recorded and the dithionite reduced minus fumarate oxidized spectra are shown; 2% correction for the dilution was made to the dithionite reduced spectrum before subtraction. The following additions were made before addition of dithionite: trace a – none; trace b – 2 μM atpenin A5, trace c – 5 μM antimycin and 10 μM myxothiazol. Trace d represents a double difference spectrum after trace b was subtracted from trace a.

Figure 7

Reoxidation of dithionite-reduced S. cerevisiae mitochondrial membranes by fumarate. The experiment was performed as described in the legend for Fig. 6. Yeast membranes were suspended at concentrations 1.8 mg protein/ml. The dashed line represents a spectrum of dithionite reduced membranes before addition of fumarate. Trace a – no additions; trace b – 2 μM atpenin A5, trace c – 5 μM antimycin and 10 μM myxothiazol.