Tài liệu Báo cáo Y học: Caged O2 Reaction of cytochrome bo3 oxidase with photochemically released

Caged O2

Reaction of cytochrome bo3 oxidase with photochemically released dioxygen

from a cobalt peroxo complex

Claudia Ludovici, Roland Fro¨ hlich*, Karsten Vogtt, Bjo¨ rn Mamat† and Mathias Lu¨ bben

Lehrstuhl fu¨r Biophysik, Ruhr-Universita¨t Bochum, Germany

We developed the synthesis of the caged oxygen donor

(l-peroxo)(l-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex

(HPBC) as nitrate salt,which has,compared with the

perchlorate-form described previously [MacArthur,R.,

Sucheta,A.,Chong,F.F. & Einarsdottir,O¨. (1995) Proc.

Natl Acad. Sci. USA, 92,8105–8109],greatly enhanced

solubility. Now,the quantum efficiency of the photolytical

release of dioxygen was determined to be 0.4 per photon at a

laser wavelength of 308 nm,which was used to observe

biological reactions. The X-ray structure of HPBC has been

solved,and the molecular interactions of photochemically

generated oxygen with cytochrome oxidase were investi￾gated with optical and FT-IR spectroscopy: it acts as

acceptor of electrons transferred from prereduced cyto￾chrome bo3,the heme-copper oxidase from Escherichia coli.

FT-IR spectra revealed typical absorbance difference chan￾ges in the carbonyl region of cytochrome bo3,supported by

bandshifts due to solvent isotope exchange and by assign￾ment using site-directed mutants. IR difference spectra of the

photooxidation reaction using the caged oxygen compound,

and of the photoreduction reaction using the caged electron

donor FMN,have inverted shapes. The spectroscopic sig￾nals of carboxyl groups are thus equivalent in both reactions:

the use of chemically produced oxygen allows the observa￾tion of the ongoing molecular changes of cytochrome bo3

oxidase under quasi-physiological conditions.

Keywords: cytochrome oxidase; caged compound; FT-IR

spectroscopy; oxygen, l-peroxo cobalt complex.

Cytochrome oxidases are hetero-oligomeric integral mem￾brane proteins that belong to the superfamily of heme￾copper oxidases [1,2]. They are terminal parts of the aerobic

respiratory chains of bacteria and mitochondria,and their

common characteristic is the transfer of electrons from

cytochrome c or ubiquinol to the acceptor substrate,

molecular dioxygen [3]. Cytochrome bo3 oxidase of

Escherichia coli is a ubiquinol oxidase. It transfers electrons

from the membrane site via heme b to the binuclear reaction

center,which consists of a heme o plus a CuB as redox

carriers. The reaction center provides the binding site of

molecular oxygen,which receives electrons and protons

necessary for water formation. The electronic energy is

sufficient to drive transmembrane proton transport,which

is tightly coupled to the processes of oxygen reduction and

of water formation [4,5].

X-ray structure data of ubiquinol oxidase from Escheri￾chia coli have been recently published. The resolution of

3.5 A˚ allows the reconstruction of the backbone but not of

the amino-acid side chain conformations [6]. Detailed

molecular structures of the cytochrome c oxidases from

Paracoccus denitrificans and beef heart mitochondria [6–10]

have been determined. Due to their extensive sequence

similarities these structures could serve as models for the

ubiquinol oxidase. They allow the prediction of two

different proton-translocating channels,called the K- and

D-channels. The D-channel contains an array of charged or

polar amino acids,and is located within two different

hydrogen-bonded networks above and below the central

Glu286 (numbering according to the subunits I and II of the

E. coli oxidase),which interacts with the binuclear center

[11]. Molecular dynamics calculations [12,13] have predicted

a special role of the central Glu286,which could provide the

contact between both partial networks. FT-IR difference

spectroscopy,using either an electrochemical cell [14,15] or

photoreduction techniques [16,17] provides information

about the orientation of amino-acid side chains and about

molecular interactions. The photoreduction experiments are

designed in such a way,that pre-equilibrated molecules

become activated by light to undergo redox changes. Out of

the many functional groups present in the oxidase,only

those affected by the redox transition become visible in

FT-IR difference spectra. In a previous report,the band

signature at 1745 cm)1 and at 1735 cm)1 occurring in redox

FT-IR difference spectra of different heme-copper oxidases

has been assigned to Glu286 [17].

In order to study the oxidase reaction with the natural

substrate dioxygen at the molecular level with FT-IR

spectroscopy,we established a caged dioxygen system that

allows O2 release via photolysis. Photoactivation of

(l-peroxo)(l-hydroxo)bis[bis(bipyridyl)cobalt(III)] complex

Correspondence to M. Lu¨bben,Lehrstuhl fu¨r Biophysik,

Ruhr-Universita¨t Bochum,Universita¨tsstr. 150,

D-44780 Bochum,Germany

Fax: + 49 234 32 14626,Tel.: + 49 234 32 24465,

E-mail: [email protected]

Abbreviations: HPBC,(l-peroxo)(l-hydroxo)bis[bis(bipyridyl)-

cobalt(III)]; BC,bis(2,2¢-bipyridyl)cobalt(II).

*Present address: Organisch-chemisches Institut,Universita¨t Mu¨nster,

Correnstraße 40,D-48149 Mu¨nster,Germany.

Present address: Max-Planck-Institut fu¨r Biophysik,

Heinrich-Hoffmann-Str. 7,D-60528 Frankfurt/Main,Germany.

(Received 18 January 2002,revised 15 April 2002,

accepted 19 April 2002)

Eur. J. Biochem. 269,2630–2637 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.02944.x