A missense mutation of cytochrome oxidase subunit II causes defective assembly and myopathy - PubMed (original) (raw)

J W Taanman, J M Cooper, I Nelson, I Hargreaves, B Meunier, M G Hanna, J J García, R A Capaldi, B D Lake, J V Leonard, A H Schapira

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A missense mutation of cytochrome oxidase subunit II causes defective assembly and myopathy

S Rahman et al. Am J Hum Genet. 1999 Oct.

Abstract

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.

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Figures

Figure 1

Figure 1

COX-activity staining (panels A and B) and COX immunohistochemical staining (panels C–H) in muscle. A = control; B = patient; C = COX subunit II (control); D = COX subunit II (patient); E = COX subunit I (patient); F = COX subunit IV (patient); G = COX subunit Va (patient); and H = COX subunit VIc (patient). Control sections stained for subunits I, IV, Va, and VIc appeared the same as section C.

Figure 2

Figure 2

Immunoblot analysis of skeletal muscle mitochondrial fractions, from the patient (P) and two controls (C). Blots were developed with subunit-specific monoclonal antibodies to COX subunits, the flavoprotein subunit of SDH (SDH Fp), core protein 1 of complex III (core 1), the α subunit of F1-ATP synthase (F1-α), and VDAC.

Figure 3

Figure 3

Flash photolysis and recombination spectra of the CO-ferroheme _a_3 compound of dissolved muscle mitochondria, from the patient and a control. After reduction by sodium dithionite and treatment with CO, laser-flash photolysis and recombination of CO was monitored at 430-445 nm versus time.

Figure 4

Figure 4

Sequence chromatogram of mitochondrial COX II gene, in control sequence (top) and patient's sequence (bottom). The arrow indicates mutated base (adenine in place of wild-type thymine).

Figure 5

Figure 5

Alignment of amino acid sequence in COX subunit II (mitochondrial genome sequence data obtained from gopher directory at the Molecular Evolution and Organelle Genomics web site). The large box represents the first α-helical region in the bovine structure (Tsukihara et al. 1996). The mutation in our patients involves amino acid residue 29.

Figure 6

Figure 6

Structure of monomer of bovine cytochrome c oxidase. The diagram shows the assembled bovine holoenzyme, with an arrow indicating the site, in subunit II, that is mutated in our patient. The diagram was constructed with use of the data published by Tsukihara et al. (1996) in the Quanta program.

References

Electronic-Database Information

    1. Molecular Evolution and Organelle Genomics, http://megasun.bch.umontreal.ca (for COX subunit II mitochondrial genome sequence data)

References

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