Tài liệu Báo cáo khoa học: Purification and characterization of a membrane-bound enzyme complex from

Purification and characterization of a membrane-bound enzyme

complex from the sulfate-reducing archaeon Archaeoglobus fulgidus

related to heterodisulfide reductase from methanogenic archaea

Gerd J. Mander1

, Evert C. Duin1

, Dietmar Linder2

, Karl O. Stetter3 and Reiner Hedderich1

1

Max-Planck-Institut fu¨r terrestrische Mikrobiologie, Marburg, Germany; 2

Biochemisches Institut, Fachbereich Humanmedizin,

Justus-Liebig-Universita¨t Giessen, Germany; 3

Lehrstuhl fu¨r Mikrobiologie und Archaeenzentrum, Universita¨t Regensburg, Germany

Heterodisulfide reductase (Hdr) is a unique disulfide reduc￾tase that plays a key role in the energy metabolism of

methanogenic archaea. The genome of the sulfate-reducing

archaeon Archaeoglobus fulgidus encodes several proteins of

unknown function with high sequence similarity to the

catalytic subunit of Hdr. Here we report on the purification

of a multisubunit membrane-bound enzyme complex from

A. fulgidus that contains a subunit related to the catalytic

subunit of Hdr. The purified enzyme is a heme/iron-sulfur

protein, as deduced by UV/Vis spectroscopy, EPR spec￾troscopy, and the primary structure. It is composed of four

different subunits encoded by a putative transcription unit

(AF499, AF501–AF503). A fifth protein (AF500) encoded

by this transcription unit could not be detected in the purified

enzyme preparation. Subunit AF502 is closely related to the

catalytic subunit HdrD of Hdr from Methanosarcina bark￾eri. AF501 encodes a membrane-integral cytochrome, and

AF500 encodes a second integral membrane protein. AF499

encodes an extracytoplasmic iron-sulfur protein, and AF503

encodes an extracytoplasmic c-type cytochrome with three

heme c-binding motifs. All of the subunits show high

sequence similarity to proteins encoded by the dsr locus of

Allochromatium vinosum and to subunits of the Hmc

complex from Desulfovibrio vulgaris. The heme groups of the

enzyme are rapidly reduced by reduced 2,3-dimethyl-1,4-

naphthoquinone (DMNH2), which indicates that the

enzyme functions as a menaquinol–acceptor oxidoreduc￾tase. The physiological electron acceptor has not yet been

identified. Redox titrations monitored by EPR spectroscopy

were carried out to characterize the iron-sulfur clusters of the

enzyme. In addition to EPR signals due to [4Fe-4S]+ clus￾ters, signals of an unusual paramagnetic species with g values

of 2.031, 1.994, and 1.951 were obtained. The paramagnetic

species could be reduced in a one-electron transfer reaction,

but could not be further oxidized, and shows EPR properties

similar to those of a paramagnetic species recently identified

in Hdr. In Hdr this paramagnetic species is specifically

induced by the substrates of the enzyme and is thought to be

an intermediate of the catalytic cycle. Hence, Hdr and the

A. fulgidus enzyme not only share sequence similarity, but

may also have a similar active site and a similar catalytic

function.

Keywords: Archaeoglobus fulgidus; heterodisulfide reductase;

Hmc complex; iron-sulfur proteins; sulfate-reducing

bacteria.

Heterodisulfide reductase (Hdr) is a key enzyme in the

energy metabolism of methanogenic archaea. In the final

step of methanogenesis, the mixed disulfide of the metha￾nogenic thiol coenzymes coenzyme M and coenzyme B is

generated in a reaction catalyzed by methyl-coenzyme M

reductase [1]. This disulfide is reduced by a unique disulfide

reductase, designated heterodisulfide reductase (Hdr). Two

types of Hdr from phylogenetically distantly related meth￾anogens have been identified and characterized. Neither

type of enzyme belongs to the family of pyridine nucleotide

disulfide oxidoreductases [2].

Hdr from Methanothermobacter marburgensis is an

iron-sulfur flavoprotein composed of the subunits HdrA,

HdrB, and HdrC. The enzyme has been purified from the

soluble fraction, and none of its subunits are predicted to

form transmembrane helices. From sequence data, it has

been deduced that HdrA contains an FAD-binding motif

and four binding motifs for [4Fe-4S] clusters. HdrC

contains two additional binding motifs for [4Fe-4S]

clusters [2].

Hdr in the two closely related Methanosarcina species

M. barkeri and M. thermophila is tightly membrane￾bound [3–5]. The enzyme is composed of two subunits,

a membrane-bound b-type cytochrome (HdrE) and a

hydrophilic subunit (HdrD) containing two binding

motifs for [4Fe-4S] clusters. Subunit HdrD of the

M. barkeri enzyme is a homologue of a hypothetical

fusion protein of the M. marburgensis HdrCB subunits

Correspondence to R. Hedderich, Max-Planck-Institut fu¨r terrestrische

Mikrobiologie, Karl-von-Frisch-Strabe, D-35043 Marburg/Germany.

Fax: + 49 6421 178299, Tel.: + 49 6421 178230,

E-mail: [email protected]

Abbreviations: Hme, Hdr-like menaquinol-oxidizing enzyme; Hdr,

heterodisulfide reductase; DMN, 2,3-dimethyl-1,4-naphthoquinone;

H-S-CoM, coenzyme M or 2-mercaptoethanesulfonate; H-S-CoB,

coenzyme B or 7-mercaptoheptanoylthreonine phosphate;

CoM-S-S-CoB, heterodisulfide of H-S-CoM and H-S-CoB; Hmc,

high-molecular-mass c-type cytochrome; Dsr, dissimilatory sulfite

reductase.

Enzyme: heterodisulfide reductase (EC 1.99.4.-).

(Received 10 October 2001, revised 12 February 2002, accepted 15

February 2002)

Eur. J. Biochem. 269, 1895–1904 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02839.x