Tài liệu Báo cáo khoa học: Enlarging the gas access channel to the active site renders the

Enlarging the gas access channel to the active site renders

the regulatory hydrogenase HupUV of Rhodobacter

capsulatus O2 sensitive without affecting its transductory

activity

Ophe´lie Duche´

1

, Sylvie Elsen1

, Laurent Cournac2 and Annette Colbeau1

1 Laboratoire de Biochimie et Biophysique des Syste`mes Inte´gre´ s (UMR 5092 CNRS-CEA-UJF), De´partement Re´ponse et Dynamique

Cellulaires, Grenoble, France

2 CEA Cadarache, De´partement des Sciences du Vivant, De´partement d’Ecophysiologie Ve´ge´tale et de Microbiologie, Laboratoire

d’Ecophysiologie de la Photosynthe` se, UMR 6191 CNRS-CEA-Aix Marseille II, Saint Paul-lez-Durance, France

Hydrogenases are enzymes involved in H2 metabolism.

They occur widely in bacteria and in some eukaryotes

[1]. The various hydrogenases differ in their metal con￾tent (FeFe, NiFe), their localization in the cell, their

relationship with metabolism, and the way their synthe￾sis is regulated [2]. They catalyze the reversible reaction

H2 « 2H++2e) and are known to be O2 sensitive. In

general, iron hydrogenases, which actively evolve H2,

are quickly and irreversibly inactivated in the presence

of O2 [3]. In contrast, most [NiFe] hydrogenases are

only reversibly inhibited by O2.

The structure of the bimetallic active site and the

mechanisms of hydrogen oxidation in [NiFe] hydro￾genases have been thoroughly studied by various bio￾physical methods (reviewed in [4,5]). The information

obtained has given clues to the inactivation of the

enzyme by O2. In Desulfovibrio hydrogenases, it has

been shown that the Fe atom is linked to three non￾protein ligands: 1 CO and 2 CN– [6]. The Ni and Fe

ions are asymmetrically bridged by two cysteine sulfur

atoms and one oxygenic species (O2

– or OH–

), which

appears in the oxidized enzyme [7–9]. The catalytic

Keywords

gas access channel; hydrogenases; oxygen

sensitivity; Rhodobacter capsulatus

Correspondence

A. Colbeau, Laboratoire de Biochimie et

Biophysique des Syste`mes Inte´gre´ s, DRDC,

CEA ⁄ Grenoble, 17 rue des martyrs,

38054 Grenoble Cedex 9, France

Fax: +33 4 38 78 51 85

Tel: +33 4 38 78 30 74

E-mail: [email protected]

Website: http://www-dsv.cea.fr/bbsi

(Received 13 May 2005, revised 26 May

2005, accepted 6 June 2005)

doi:10.1111/j.1742-4658.2005.04806.x

In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of

the energy-producing hydrogenase, HupSL, is regulated by the substrate

H2, which is detected by a regulatory hydrogenase, HupUV. The HupUV

protein exhibits typical features of [NiFe] hydrogenases but, interestingly,

is resistant to inactivation by O2. Understanding the O2 resistance of

HupUV will help in the design of hydrogenases with high potential for bio￾technological applications. To test whether this property results from O2

inaccessibility to the active site, we introduced two mutations in order to

enlarge the gas access channel in the HupUV protein. We showed that such

mutations (Ile65 fi Val and Phe113 fi Leu in HupV) rendered HupUV

sensitive to O2 inactivation. Also, in contrast with the wild-type protein,

the mutated protein exhibited an increase in hydrogenase activity after

reductive activation in the presence of reduced methyl viologen (up to 30%

of the activity of the wild-type). The H2-sensing HupUV protein is the first

component of the H2-transduction cascade, which, together with the two￾component system HupT⁄ HupR, regulates HupSL synthesis in response to

H2 availability. In vitro, the purified mutant HupUV protein was able to

interact with the histidine kinase HupT. In vivo, the mutant protein exhib￾ited the same hydrogenase activity as the wild-type enzyme and was equally

able to repress HupSL synthesis in the absence of H2.

Abbreviations

MG medium, malate ⁄ glutamate medium; MN medium, malate ⁄ ammonia medium; RH, regulatory hydrogenase; SH, soluble NAD-linked

hydrogenase.

FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS 3899