Tài liệu Báo cáo Y học: Mutations in the docking site for cytochrome c on the Paracoccus heme aa3

Mutations in the docking site for cytochrome c on the Paracoccus

heme aa3 oxidase

Electron entry and kinetic phases of the reaction

Viktoria Drosou1

, Francesco Malatesta2 and Bernd Ludwig1

1

Molecular Genetics, Institute of Biochemistry, Johann-Wolfgang-Goethe Universita¨t, Frankfurt, Germany; 2

Department of Basic and Applied Biology, University of L’Aquila, Italy

Introducing site-directed mutations in surface-exposed

residues of subunit II of the heme aa3 cytochrome c oxidase

of Paracoccus denitrificans, we analyze the kinetic para￾meters of electron transfer from reduced horse heart cyto￾chrome c. Specifically we address the following issues: (a)

which residues on oxidase contribute to the docking site for

cytochrome c, (b) is an aromatic side chain required for

electron entry from cytochrome c, and (c) what is the

molecular basis for the previously observed biphasic reaction

kinetics. From our data we conclude that tryptophan 121 on

subunit II is the sole entry point for electrons on their way to

the CuA center and that its precise spatial arrangement, but

not its aromatic nature, is a prerequisite for efficient electron

transfer. With different reaction partners and experimental

conditions, biphasicity can always be induced and is critically

dependent on the ionic strength during the reaction. For an

alternative explanation to account for this phenomenon, we

find no evidence for a second cytochrome c binding site on

oxidase.

Keywords: Paracoccus denitrificans; cytochrome c oxidase;

docking site; electron transfer; biphasic kinetics.

Cytochrome c oxidase is the terminal complex of the

respiratory chains of mitochondria and many bacteria

[1–4]. It catalyzes the reduction of oxygen to water, coupling

the free energy of this reaction to the generation of a proton

gradient across the membrane.

During the redox reaction, an electron delivered from

cytochrome c is first transferred to CuA, a binuclear copper

center located close to the surface of the large hydrophilic

domain of subunit II. It is then donated to heme a

embedded in subunit I, and subsequently to the heme a3Æ

CuB center where oxygen reduction, and most likely the

redox coupling to proton pumping, take place.

While the mitochondrial enzyme comprises up to 13

different subunits in a dimeric complex, the oxidase of the

bacterium Paracoccus denitrificans consists of only four

subunits, with the three largest ones homologous to the

corresponding mitochondrial subunits.

Typically, many isolated oxidases are somewhat promis￾cuous towards their substrate molecules. Early studies

analyzing the surface properties of cytochromes c of

different origin revealed a basic cluster of mostly lysine

residues located around the heme crevice. Being responsible

for docking to their redox partners, an interaction between

cytochrome c and oxidase based on electrostatic forces was

described [5–7]. Experiments with monoclonal antibodies

directed against subunit II of cytochrome c oxidase lead to a

loss of activity [8] and supported the notion that the catalytic

binding site is located predominantly on subunit II. This

result was confirmed by chemical modifications and early

site-directed mutagenesis experiments [9,10], and is consis￾tent with the crystal structures of the eukaryotic and the

Paracoccus denitrificans oxidase showing clusters of negat￾ively charged residues on the surface of subunit II [11,12].

Previous studies on the binding of cytochrome c to the

Paracoccus oxidase were interpreted by a two-step model in

which electrostatic forces are responsible for an efficient

long-range docking, followed by the reorientation of the

redox partner driven by hydrophobic interactions [13].

Specifically, a set of four acidic residues exposed on subunit

II (D135, D178, and to a lesser extent, E126, D159; see

Fig. 1 and [13]) had been assumed to interact electrostat￾ically with the horse heart cytochrome c. While a pivotal

role in electron transfer from cytochrome c was assigned to

residue W121 on subunit II [14], this early study did not

address the question whether any other (aromatic) side

chains in this or the neighbouring position might be able to

support electron transfer to the CuA site, thereby function￾ally replacing tryptophan 121.

As already observed previously, oxidase kinetics may

yield nonlinear Eadie–Hofstee plots (e.g. [15]). Two different

phases, denoted high and low affinity, are clearly discern￾ible, each being characterized by a set of individual kinetic

parameters. These biphasic steady-state kinetics become

monophasic at higher ionic strength, a phenomenon

discussed in terms of different binding sites (e.g. [15,16]).

or of conformational changes within the enzyme-substrate

complex related to the coupling of electron transfer with

proton pumping [17].

Correspondence to B. Ludwig, Molecular Genetics, Institute of

Biochemistry, Biozentrum, Marie-Curie-Strasse 9,

D-60439 Frankfurt, Germany.

Fax: + 49 69 798 29244, Tel.: + 49 69 798 29237,

E-mail: [email protected]

Abbreviations: I, ionic strength; c552-f: soluble fragment of the

Paracoccus denitrificans cytochrome c552, expressed and purified from

Escherichia coli.

(Received 4 February 2002, revised 12 April 2002,

accepted 2 May 2002)

Eur. J. Biochem. 269, 2980–2988 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.02979.x