Tài liệu Báo cáo Y học: Electrochemical, FT-IR and UV/VIS spectroscopic properties of the caa3

Electrochemical, FT-IR and UV/VIS spectroscopic properties of the caa3

oxidase from T. thermophilus

Petra Hellwig1

, Tewfik Soulimane2

* and Werner Ma¨ ntele1

1

Institut fu¨r Biophysik der Johann-Wolfgang-Goethe-Universita¨t, Frankfurt/M., Germany; 2

Institut fu¨r Biochemie der Rheinisch￾Westfa¨lischen-Technischen Hochschule, Aachen, Germany

The caa3-oxidase from Thermus thermophilus has been

studied with a combined electrochemical, UV/VIS and

Fourier-transform infrared (FT-IR) spectroscopic

approach. In this oxidase the electron donor, cytochrome c,

is covalently bound to subunit II of the cytochrome c

oxidase. Oxidative electrochemical redox titrations in the

visible spectral range yielded a midpoint potential of

)0.01 ± 0.01 V (vs. Ag/AgCl/3M KCl, 0.218 V vs. SHE¢)

for the heme c. This potential differs for about 50 mV from

the midpoint potential of isolated cytochrome c, indicating

the possible shifts of the cytochrome c potential when bound

to cytochrome c oxidase. For the signals where the hemes a

and a3 contribute, three potentials, ¼ )0.075 V ± 0.01 V,

Em2 ¼ 0.04 V ± 0.01 V and Em3 ¼ 0.17 V ± 0.02 V

(0.133, 0.248 and 0.378 V vs. SHE¢, respectively) could be

obtained. Potential titrations after addition of the inhibitor

cyanide yielded a midpoint potential of )0.22 V ± 0.01 V

for heme a3-CN– and of Em2 ¼ 0.00 V ± 0.02 V and

Em3 ¼ 0.17 V ± 0.02 V for heme a ()0.012 V, 0.208 V

and 0.378 V vs. SHE¢, respectively). The three phases of the

potential-dependent development of the difference signals

can be attributed to the cooperativity between the hemes a,

a3 and the CuB center, showing typical behavior for cyto￾chrome c oxidases. A stronger cooperativity of CuB is dis￾cussed to reflect the modulation of the enzyme to the

different key residues involved in proton pumping. We thus

studied the FT-IRspectroscopic properties of this enzyme to

identify alternative protonatable sites. The vibrational

modes of a protonated aspartic or glutamic acid at

1714 cm)1 concomitant with the reduced form of the protein

can be identified, a mode which is not present for other

cytochrome c oxidases. Furthermore modes at positions

characteristic for tyrosine vibrations have been identified.

Electrochemically induced FT-IRdifference spectra after

inhibition of the sample with cyanide allows assigning the

formyl signals upon characteristic shifts of the m(C¼O)

modes, which reflect the high degree of similarity of heme a3

to other typical heme copper oxidases. A comparison with

previously studied cytochrome c oxidases is presented and

on this basis the contributions of the reorganization of the

polypeptide backbone, of individual amino acids and of the

hemes c, a and a3 upon electron transfer to/from the redox

active centers discussed.

Keywords: caa3 oxidase; cytochrome c oxidase; UV/VIS￾spectroscopy; FT-IR-spectroscopy; Thermus thermophilus.

Cytochrome c oxidase is the terminal enzyme of the

respiratory chain in mitochondria and many prokaryotes.

As an integral membrane protein it catalyzes the reduction

of dioxygen to water using electrons from cytochrome c.

Four redox-active sites are involved in the electron transfer.

Electrons from cytochrome c are first transferred to a

homobinuclear copper A site (CuA) and then subsequently

to heme a, and to heme a3, which is located close to copper

B (CuB), forming a heterobinuclear metal center where

oxygen is reduced to water. Protons needed for water

formation are taken up from the cytosolic side in bacterial

membranes or from the matrix side in mitochondria. The

proton consumption and the coupled translocation of

n H+/e– across the membrane contribute to the proton

gradient needed to synthesize ATP.

Two pathways have been proposed to serve for consumed

and pumped protons on the basis of site-directed mutagen￾esis [1,2] and later using the crystal structures [3–5]. These

pathways are highly conserved among most studied cyto￾chrome oxidases [2,6]. However, cytochrome oxidases have

been reported that lack amino acids disputed to be essential

in proton translocation. In the case of caa3-oxidases from

T. thermophilus, for example, as well as from Rhodothermus

marinus, the amino acid Glu278 (numbering for Paracoccus

denitrificans), which is proposed to pass protons in the

D-pathway to the binuclear center, is missing, but proton￾pumping activity is observed [3,7–9]. A highly conserved

Tyr–Ser couple was suggested to replace Glu278 [9]. In the

current understanding, two pathways are necessary for the

catalytic activity, but different residues may be involved. In

an important step for the understanding of the essentials for

cytochrome c oxidase activity and coupled proton pump￾ing, the crystal structure of the aberrant ba3-oxidase from

T. thermophilus was determined [10] and alternative path￾ways discussed.

Correspondance to P. Hellwig, Institut fu¨r Biophysik der Johann￾Wolfgang-Goethe-Universita¨t, Theodor-Stern-Kai 7 Haus 74,

60590 Frankfurt/M., Germany.

Fax: + 49 69 6301 5838, Tel.: + 49 69 6301 4227,

E-mail: [email protected]

Abbreviations: FT-IR, Fourier-transform infrared; SHE¢, standard

hydrogen electrode; TMPD, N,N,N¢,N¢-tetramethyl-p-phenylenedi￾amine dihydrochloride

*Present address: Paul Scherrer Institut, Structural Biology Group,

5232-CH, Villigen PSI, Switzerland.

(Received 13March 2002, revised 6 August 2002, accepted 14 August 2002)

Eur. J. Biochem. 269, 4830–4838 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03182.x