Tài liệu Báo cáo khoa học: Reconstitution of coupled fumarate respiration in liposomes by

Reconstitution of coupled fumarate respiration in liposomes

by incorporating the electron transport enzymes isolated

from Wolinella succinogenes

Simone Biel1

, Jo¨ rg Simon1

, Roland Gross1

, Teresa Ruiz2

, Maarten Ruitenberg3 and Achim Kro¨ ger1

1

Institut fu¨r Mikrobiologie, Johann Wolfgang Goethe-Universita¨t, Frankfurt am Main, Germany; 2

Max-Planck-Institut fu¨r Biophysik,

Abteilung Strukturbiologie, Frankfurt am Main, Germany; 3

Max-Planck-Institut fu¨r Biophysik, Abteilung Biophysikalische Chemie,

Frankfurt am Main, Germany

Hydrogenase and fumarate reductase isolated from Woli￾nella succinogenes were incorporated into liposomes con￾taining menaquinone. The two enzymes were found to be

oriented solely to the outside of the resulting proteolipo￾somes. The proteoliposomes catalyzed fumarate reduction

by H2 which generated an electrical proton potential (Dw ¼

0.19 V, negative inside) in the same direction as that gen￾erated by fumarate respiration in cells of W. succinogenes.

The H+/e ratio brought about by fumarate reduction with

H2 in proteoliposomes in the presence of valinomycin and

external K+ was approximately 1. The same Dw and H+/e

ratio was associated with the reduction of 2,3-dimethyl-1,4-

naphthoquinone (DMN) by H2 in proteoliposomes con￾taining menaquinone and hydrogenase with or without

fumarate reductase. Proteoliposomes containing menaqui￾none and fumarate reductase with or without hydrogenase

catalyzed fumarate reduction by DMNH2 which did not

generate a Dw. Incorporation of formate dehydrogenase

together with fumarate reductase and menaquinone

resulted in proteoliposomes catalyzing the reduction of

fumarate or DMN by formate. Both reactions generated a

Dw of 0.13 V (negative inside). The H+/e ratio of formate

oxidation by menaquinone or DMN was close to 1. The

results demonstrate for the first time that coupled fumarate

respiration can be restored in liposomes using the well

characterized electron transport enzymes isolated from

W. succinogenes. The results support the view that Dw

generation is coupled to menaquinone reduction by H2 or

formate, but not to menaquinol oxidation by fumarate. Dw

generation is probably caused by proton uptake from the

cytoplasmic side of the membrane during menaquinone

reduction, and by the coupled release of protons from H2 or

formate oxidation on the periplasmic side. This mechanism

is supported by the properties of two hydrogenase mutants

of W. succinogenes which indicate that the site of quinone

reduction is close to the cytoplasmic surface of the

membrane.

Keywords: fumarate respiration; Wolinella succinogenes;

proteoliposomes; H+/ e ratio; hydrogenase.

The electron transport chain catalyzing fumarate respiration

with H2 (reaction a) or formate (reaction b) in Wolinella

succinogenes consists of fumarate reductase, menaquinone

(MK), and either hydrogenase or formate dehydrogenase

(Fig. 1).

H2 þ Fumarate ! Succinate ðaÞ

HCOÿ

2 þ Fumarate þ H2O ! HCOÿ

3 þ Succinate ðbÞ

The enzymes were isolated and the corresponding genes

were sequenced [2,3]. Each of the three enzymes consists of

two hydrophilic subunits and a di-heme cytochrome b

which is integrated in the membrane [4–7]. The iron–sulfur

subunits (HydA, FdhB, FrdB) mediate electron transfer

from the catalytic subunits to the cytochromes b or vice

versa [8]. The di-heme cytochromes b of hydrogenase and of

formate dehydrogenase carry the sites of MK reduction,

and are similar in their sequences [6,9,10]. Menaquinol

(MKH2) is oxidized at the di-heme cytochrome b of

fumarate reductase [4,5].

The dehydrogenases (hydrogenase and formate dehy￾drogenase) catalyze the reduction of the water soluble

MK analogue 2,3-dimethyl-1,4-naphthoquinone (DMN)

by their respective substrates (reaction c and d). The site

of DMN reduction is located on HydC [6]. Fumarate

reductase catalyzes DMNH2 oxidation by fumarate

(reaction e). The site of DMNH2 oxidation is located

on FrdC [4].

H2 þ DMN ! DMNH2 ðcÞ

Correspondence to A. Kro¨ger, Institut fu¨r Mikrobiologie, Johann

Wolfgang Goethe-Universita¨t,Marie-Curie-Str. 9, D-60439 Frankfurt

am Main, Germany.

Fax: + 49 69 79829527, Tel.: + 49 69 79829507,

E-mail: [email protected]

Abbreviations: DMN, 2,3-dimethyl-1,4-naphthoquinone; DMNH2,

hydroquinone of DMN; FCCP, carbonyl cyanide p-tri￾fluoromethoxyphenylhydrazone; FdhA/B/C, formate dehydrogenase;

FrdA/B/C, fumarate reductase; HQNO, 2-(n-heptyl)-4-hydroxyquin￾oline-N-oxide; HydA/B/C, hydrogenase A/B/C of W. succinogenes;

MK, menaquinone; MKH2, hydroquinone of MK; methyl-MK, 5- or

8-methyl-MK; TAME, N-a-tosyl-L-arginyl-O-methylester; TPP+,

tetraphenylphosphonium; TPB–

, tetraphenylboranate; Dp, electro￾chemical proton potential (proton motive force) across a membrane

(in volts); Dw, electrical proton potential across a membrane (in volts).

(Received 6 December 2001, revised 12 February 2002, accepted 21

February 2002)

Eur. J. Biochem. 269, 1974–1983 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02842.x