Structure, Mechanism and Physiological Roles of Bacterial Cytochrome c Peroxidases

John M. Atack; David J. Kelly    Department of Molecular Biology and Biotechnology, The University of Sheffield, Western Bank, Sheffield S10 2TN, UK

Abbreviations

bCCP bacterial cytochrome-c peroxidase

CCP cytochrome-c peroxidase

FDH formate dehydrogenase

hp high potential

lp low potential

MADH methylamine dehydrogenase

TTQ tryptophan tryptophylquinone

1 Introduction: enzymic mechanisms to combat oxidative and peroxidative stress

Any organism, and particularly one that uses oxygen as a terminal electron acceptor, is subject to oxidative stress. Oxidative stress results from the formation of toxic oxygen intermediates formed by incomplete reduction of oxygen. Reduction of oxygen to water is a four electron reaction, but toxic oxygen intermediates formed by incomplete reduction include the superoxide anion (O2−), hydrogen peroxide (H2O2), and the hydroxyl radical (HO·) (Storz and Imlay, 1999). Superoxide and H2O2 interact to form the highly toxic hydroxyl radical. Build up of these highly reactive species can lead to damage to proteins, nucleic acids and membranes. Reactive oxygen species are also produced by the immune system to kill invading microorganisms. Therefore, the ability to combat these toxic compounds is key to survival in the environment and the host (Storz and Imlay, 1999), and microbes have evolved an impressive array of enzyme systems to detoxify reactive oxygen species.

H2O2 is not an abundant molecule in many environments. It is formed naturally mainly by the action of sunlight on water and is thus found in traces in natural water bodies, rain and snow. H2O2 as a molecule is fairly weakly reactive, but the single bond between the two oxygen atoms is easily broken, so that it readily fragments into a hydrogen and a hydroperoxyl radical or into two hydroxyl radicals. H2O2 is generated both endogenously within cells that use oxygen as a terminal electron acceptor and exogenously by, for example, certain types of host cells when pathogens elicit an immune response (Storz and Imlay, 1999; Imlay, 2003). H2O2 can damage proteins through oxidation of co-factors such as iron–sulphur clusters (Imlay, 2002), membranes and DNA, and can react with superoxide (O2−) to form the hydroxyl radical (HO·) using iron via the Fenton reaction (Imlay et al., 1988). H2O2 is also produced as an intermediate in superoxide breakdown by superoxide dismutase (SOD), which is a major defence enzyme against damage caused by superoxide. Using two protons, SOD converts two superoxide anions to a molecule of H2O2 and a molecule of oxygen. SOD is widely distributed in nature – nearly all aerobes possess the enzyme, but it is present in only some anaerobes (Imlay, 2002, 2003).

The major sources of endogenously produced H2O2 have traditionally been thought to be respiratory chain redox enzymes, especially flavoproteins, that react with molecular oxygen to produce H2O2, but recent studies using mutants of Eschericia coli suggest that this may not be the case. Seaver and Imlay (2004) constructed an E. coli strain lacking both catalases and alkyl-hydroperoxide reductase. This strain released accumulated H2O2 into the medium, where it could be measured. Further mutations were made in this background and, surprisingly, mutants lacking either or both NADH dehydrogenase I or II and/or fumarate reductase continued to produce H2O2 at normal rates. The conclusion is that, in E. coli at least, most peroxide is generated outside the respiratory chain, but as yet, the sources of endogenous H2O2 remain to be elucidated (Seaver and Imlay, 2004). Nevertheless, irrespective of the source, bacteria possess three major types of enzymes to remove H2O2, of which the cytochrome-c peroxidases are the particular focus of this review.

1.1 Catalase

Catalase removes H2O2 from cells by splitting it to water and oxygen. Catalases can be divided into three types; mono-functional catalases, bi-functional catalase–peroxidases and manganese-containing (non-haem) catalases (Chelikani et al., 2004). Many micro-organisms contain more than one type of catalase, these often being induced under different stress conditions by the action of redox-sensing regulatory systems, e.g. OxyR in E. coli (Zheng and Storz, 2000). Although catalase is the most-widely studied H2O2 detoxification mechanism in the cytoplasm (Storz and Imlay, 1999; Chelikani et al., 2004), it may not represent the major way in which H2O2 is removed by bacteria at low concentrations (Seaver and Imlay, 2001).

1.2 Glutathione, Thioredoxin and NADH-linked Peroxidases

Glutathione peroxidases (Gpx) are selenoenzymes, which catalyse the reduction of hydroperoxides (H2O2 or ROOH) in the presence of glutathione (GSH). Although glutathione peroxidases are widespread among eukaryotes where they function as a major peroxide defence, they have not been well studied among prokaryotes. However, evidence from mutant studies in Neisseria meningitidis (Moore and Sparling, 1996) and Streptococcus pyogenes (Brenot et al., 2004) indicate a role in the protection against H2O2, and in the latter case a role in virulence.

Alkyl-hydroperoxide reductase (Ahp) and the related proteins thiol-peroxidase (Tpx) and “bacterioferritin-comigratory protein” (Bcp) are non-haem peroxidases, and are members of the peroxiredoxin family (Poole et al., 2000; Poole, 2005). These enzymes reduce reactive hydroperoxides to their corresponding alcohols using thioredoxin or NADH as an electron donor, and can also reduce H2O2 (Poole et al., 2000; Baker et al., 2001; Guimaraes et al., 2005). In fact, mutant studies have shown that in E. coli, Ahp is more important than catalase in removing low concentrations of H2O2, due to a higher affinity (Seaver and Imlay, 2001). AhpC has also been shown to be a peroxynitrite reductase (Bryk et al., 2000). Hydroperoxides are a problem in cells as they are able to initiate and propagate free radical chain reactions that can cause significant damage to DNA and membranes (Baillon et al., 1999). In E. coli, Ahp consists of two subunits. AhpC contains the catalytic site, and the flavoprotein AhpF is the electron donor to AhpC (Poole et al., 2000). AhpC homologues are found in many organisms, and all contain highly conserved cys residues at their N- and C-termini (Baillon et al., 1999). Activity was shown to be dependent on the N-terminal cysteine (Ellis and Poole, 1997; Bryk et al., 2000) with the C-terminal cysteine stabilising the oxidised protein via an intersubunit interaction (Ellis and Poole, 1997). In catalysis, the N-terminal cysteine is oxidised to a cys-sulphenic acid upon addition of the hydroperoxide substrate, with the second cysteine stabilising this via disulphide bond formation (Wood et al., 2003). Like catalase, AhpC is cytoplasmic. In many organisms, AhpC exists as an oligomer, usually as a pentamer or hexamer of dimers (Guimaraes et al., 2005; Parsonage et al., 2005). In Helicobacter pylori, mutation of ahpC led to a significant decrease in catalase activity. This is due to the disruption of the haem environment of catalase by an increase in organic peroxides in the cell. Thus, AhpC prevents catalase inactivation by removing these organic peroxides (Wang et al., 2004). It is not clear if AhpC is an important virulence factor. Cells of Porphyromonas gingivalis, an aetiological agent of periodontitis, lacking AhpC were more sensitive to organic peroxides, but were just as virulent as wild-type cells in the mouse model of infection (Johnson et al., 2004). Campylobacter jejuni mutants lacking ahpC were hypersensitive to killing by cumene...