Tài liệu Báo cáo khoa học: NirF is a periplasmic protein that binds d1 heme as part of its essential

NirF is a periplasmic protein that binds d1 heme as part of

its essential role in d1 heme biogenesis

Shilpa Bali1

, Martin J. Warren2 and Stuart J. Ferguson1

1 Department of Biochemistry, University of Oxford, UK

2 Department of Biosciences, University of Kent, Canterbury, UK

Introduction

Denitrification is a four-step transformation of nitrate

to dinitrogen gas by various species of bacteria under

anaerobic conditions [1,2]. These four steps are cataly￾sed by complex metalloenzymes and involve stepwise

conversion of nitrate to nitrite, nitrite to nitric oxide,

nitric oxide to nitrous oxide and finally reduction of

nitrous oxide to nitrogen. In the denitrification path￾way, nitrite reduction is the key step, as it is the point

of divergence from assimilatory nitrogen metabolism in

which nitrite is reduced to ammonium [2,3]. There are

two types of respiratory nitrite reductase involved in

denitrification: one is copper-containing nitrite reduc￾tase (NirK), which is prevalent in, but not exclusive to,

alphaproteobacteria, the other being cytochrome cd1

(NirS), which prevails in betaproteobacteria [4].

Cytochrome cd1 nitrite reductase is a homodimeric

periplasmic enzyme with each subunit containing a

covalently attached c heme and noncovalently attached

d1 heme, bound in a beta-propeller domain, as pros￾thetic groups [5,6]. Heme d1, which forms the active cen￾tre for the one electron reduction of nitrite to nitric

oxide, has a unique structure. The structure of this mod￾ified heme, a dioxoisobacteriochlorin to be more spe￾cific, has been known for more than two decades [7,8],

but quite how it is biosynthesized by denitrifying bacte￾ria under anaerobic conditions is not understood. Anal￾ysis of insertional mutagenesis and complementation

work in Pseudomonas aeruginosa, Pseudomonas fluores￾cens, Paracoccus denitrificans and Pseudomonas stutzeri

have shown that a set of several contiguous genes that

always follows the structural gene, nirS, for cytochrome

cd1, is necessary for the biogenesis of the d1 cofactor

[9–13]. In P. denitrificans and closely related Para￾coccus pantotrophus, these genes are cotranscribed as

Keywords

cytochrome cd1; d1 heme biosynthesis;

denitrification; nitrite reductase;

Paracoccus pantotrophus; tetrapyrrole

Correspondence

S. J. Ferguson, Department of

Biochemistry, University of Oxford, South

Parks Road, Oxford OX1 3QU, UK

Fax: +44 1865 613201

Tel: +44 1865 613299

E-mail: [email protected]

(Received 24 June 2010, revised 27 August

2010, accepted 1 October 2010)

doi:10.1111/j.1742-4658.2010.07899.x

The cytochrome cd1 nitrite reductase from Paracoccus pantotrophus catalyses

the one electron reduction of nitrite to nitric oxide using two heme cofactors.

The site of nitrite reduction is the d1 heme, which is synthesized under anaer￾obic conditions by using nirECFD-LGHJN gene products. In vivo studies

with an unmarked deletion strain, DnirF, showed that this gene is essential

for cd1 assembly and consequently for denitrification, which was restored

when the DnirF strain was complemented with wild-type, plasmid-borne,

nirF. Removal of a signal sequence and deletion of a conserved N-terminal

Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo

NirF activity. We demonstrate here that the product of the nirF gene is a

periplasmic protein and, hence, must be involved in a late stage of the cofac￾tor biosynthesis. In vitro studies with purified NirF established that it could

bind d1 heme. It is concluded that His41 of NirF, which aligns with His200

of the d1 heme domain of cd1, is essential both for this binding and for the

production of d1 heme; replacement of His41 by Ala, Cys, Lys and Met all

gave nonfunctional proteins. Potential functions of NirF are discussed.

Abbreviation

LB, Luria–Bertani.

4944 FEBS Journal 277 (2010) 4944–4955 ª 2010 The Authors Journal compilation ª 2010 FEBS