Tài liệu Báo cáo khoa học: Adaptive changes in the expression of nuclear and mitochondrial encoded

Adaptive changes in the expression of nuclear and mitochondrial

encoded subunits of cytochrome c oxidase and the catalytic

activity during hypoxia

C. Vijayasarathy1,*,†, Shirish Damle1,*, Subbuswamy K. Prabu1,*, Cynthia M. Otto2

and Narayan G. Avadhani1

1

Department of Animal Biology and 2

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania,

Philadelphia, PA, USA

The effects of physiologically relevant hypoxia on the

catalytic activity of cytochrome c oxidase (CytOX), mito￾chondrial gene expression, and both nuclear and mito￾chondrial encoded CytOX mRNA levels were investigated

in murine monocyte macrophages, mouse C2C12 skeletal

myocytes and rat adrenal pheochromocytoma PC12 cells.

Our results suggest a coordinated down regulation of mito￾chondrial genome-coded CytOX I and II and nuclear

genome-coded CytOX IV and Vb mRNAs during hypoxia.

Hypoxia also caused a severe decrease in mitochondrial

transcription rates, and associated decrease in mitochondrial

transcription factor A. The enzyme from hypoxia exposed

cells exhibited altered subunit content as revealed by blue

native gel electrophoresis. There was a generalized decline in

mitochondrial function that led to a decrease in total cellular

heme and ATP pools. We also observed a decrease in

mitochondrial heme aa3 content and decreased levels of

CytOX subunit I, IV and Vb, though the catalytic efficiency

of the enzyme (TN for cytochrome c oxidase) remained

nearly the same. Increased glycolytic flux and alterations in

the kinetic characteristics of the CytOX might be the two

mechanisms by which hypoxic cells maintain adequate ATP

levels to sustain life processes. Reoxygenation nearly com￾pletely reversed hypoxia-mediated changes in CytOX

mRNA contents, rate of mitochondrial transcription, and

the catalytic activity of CytOX enzyme. Our results show

adaptive changes in CytOX structure and activity during

physiological hypoxia.

Keywords: hypoxia; cytochrome c oxidase; subunit content;

mitochondrial genome transcription.

Cytochrome c oxidase (CytOX) is the terminal oxidase of

the mitochondrial electron transport chain [1–5], which

catalyzes the reduction of the dioxygen (O2) to water and

harnesses the free energy of the reaction to phosphorylate

ADP to ATP. Heme and Cu, which transfer electrons from

ferrocytochrome c to molecular oxygen, constitute the

catalytic site of the enzyme complex. The three catalytic

subunits, CytOX I, II and III are coded by the mitochon￾drial DNA and are synthesized within mitochondria.

Heme a, heme a3 and Cub are ligated to subunit I, while

Cua is ligated to subunit II which is also the binding site for

cytochrome c [4,5]. The remaining 10 subunits of the

mammalian enzyme, namely, IV, Vb, VIa, VIb, VIc, VIIa,

VIIb and VIII are encoded by the nuclear genome,

synthesized in the cytosol and imported into mitochondria

[1–3]. Some of the nuclear-encoded subunits in the mam￾mals are regulated developmentally and occur as tissue

specific isoforms [6,7]. Although the nuclear encoded

subunits, such as CytOX VIa and VIb, have been shown

to enhance the catalytic efficiency of the enzyme [8,9], the

precise role of many nuclear-encoded subunits in the

mammalian enzyme complex remains unknown.

Oxygen as a substrate and heme as a prosthetic group, are

closely interlinked in the function of the enzyme complex.

Studies in yeast have shown that both oxygen and heme act

as physiological modulators and regulate the expression of

the enzyme complex [10]. In the yeast CytOX complex, the

nuclear encoded subunit V is expressed as two distinct

isoforms, Va and Vb, that are regulated by heme and O2 [11].

In the mammalian systems, however, the differential expres￾sion of nuclear encoded subunits in response to different

physiological factors has not been investigated in detail.

In a previous study we demonstrated that administration

of inhibitors of heme biosynthesis (succinyl acetone and

cobalt chloride) to mice, resulted in a 50% reduction in

mitochondrial genome encoded CytOX I and II mRNAs

and nuclear genome encoded CytOX Vb mRNA in heme

depleted tissues [12]. Heme depletion was also accom￾panied by a 50–80% reduction in intramitochondrial

Correspondence to N. G. Avadhani, Department of Animal Biology,

School of Veterinary Medicine, University of Pennsylvania,

3800 Spruce Street, Philadelphia, PA, 19104, USA.

Fax: + 215 573 6651, Tel.: + 215 898 8819,

E-mail: [email protected]

Abbreviations: CytOX, cytochrome c oxidase; mtTFA, mitochondrial

transcription factor A; SMP, submitochondrial particles; TN, turn￾over number; BN/PAGE, blue native gel electrophoresis.

*Note: these authors contributed equally to this work.

Present address: UAE University, Faculty of Medicine and Health

Sciences, Department of Biochemistry, Al Ain, United Arab Emirates.

(Received 8 November 2002, revised 18 December 2002,

accepted 3 January 2003)

Eur. J. Biochem. 270, 871–879 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03447.x