Iron Sulfur Proteins in Organisms Harboring Hydrogenosomes and Mitosomes

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The common feature of organisms with hydrogenosomes or mitosomes is that they inhabit oxygen-poor environments. Their energy metabolism is fermentative, producing pyruvate via a classic Embden-Mayerhof pathway; however, the further metabolism of pyruvate, a key intermediate product, which is linked to ATP production by substrate-level phosphorylation only, is significantly different. Consequently, a different set of FeS proteins is involved in this process. These proteins are compartmentalized either into hydrogenosemes (Trichomonas, Neocalimastix) or into the cytosol (Entamoeba, Giardia, Cryptosporidium, microsporidia) (Hackstein et al. 1999; Katinka et al. 2001; Müller 1988, 2003; Xu et al. 2004; Table 6.1).

In trichomonad hydrogenosomes, pyruvate is oxidatively decarboxylated by [4Fe4S] pyruvate:ferredoxin oxidoreductase (PFO). Released electrons are transferred via [2Fe2S] ferredoxin to [Fe]-hydrogenase, producing H2. In contrast, in fungal hydrogenosomes, pyruvate is cleaved by pyruvate: formate lyase, in which ferredoxin is not involved. Malate is another hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate by NAD(P)-dependent malic enzyme. In trichomonads, the transfer of electrons released during this reaction from NADH to ferredoxin can be monitored as NADH:ferredoxin or NADH: methylviologen oxidoreductase

Table 6.1. Distrubution of FeS proteins in organisms with hydrogenosomes or mitosomes

PFO puruvate: ferredoxin oxidoreductase, HCP hybrid cluster protein, PNO

Trichomonas vaginalis

Giardia intestinalis

Entamoeba histolytica

Cryptosporidium parvum

Encephalitozoon cuniculi

Hydrogenosomes/ mitosomes

Hydrogenase

[2Fe-2S] ferredoxin

[2Fe-2S] ferredoxin

[2Fe-2S] ferredoxin

[2Fe-2S] ferredoxin

Tvh47(complex I)

Tvh22(complex I)

IscU

IscU

IscU

IscU

I sc A

IscA

FeS flavoprotein

Cytosol

HCP

Hydrogenase? [4Fe-4S] ferredoxin [3Fe-4S] ferredoxin

HCP

FeS flavoprotein Hydrogenase? [4Fe-4S] ferredoxin [3Fe-4S] ferredoxin NifU

PNO

Rli

Rli

Rli

Rli

Rli

Nbp35

Nbp35

Nbp35

Nbp35

Nbp35

Nar/Narf

Nar/Narf

Nar/Narf

Nar/Narf

Nar/Narf

Nucleus

DNA glycosylase

DNA glycosylase

DNA glycosylase

DNA glycosylase

PFO puruvate:ferredoxin oxidoreductase, HCP hybrid cluster protein, PNO pyruvate:NADH oxidoreductase, Nbp35 P-loop NTPase, Nnr/Nnr/hydrogenase-like protein, Rli ATP-binding cassette protein

PFO puruvate:ferredoxin oxidoreductase, HCP hybrid cluster protein, PNO pyruvate:NADH oxidoreductase, Nbp35 P-loop NTPase, Nnr/Nnr/hydrogenase-like protein, Rli ATP-binding cassette protein activity (Steinbühel and Müller 1986; Thong and Coombs 1987). Recently it was shown that this step of electron transport is catalyzed by het-erodimeric NADH dehydrogenase with homology to the promotory module of mitochondrial respiratory complex I (Hrdy et al. 2004). In mitochondria, NADH dehydrogenase is a membrane-bound multi-sub-unit protein complex, which is composed of up to 43 subunits (Yano 2002). It catalyzes transfer of electrons from NADH to the lipid-soluble electron carrier ubiquinone, which is coupled to the proton translocation. It is organized into two parts: a hydrophilic (promotory) part, containing most of the redox cofactors, and a hydrophobic (proton-pumping) part that is anchored in the membrane. The promotory part contains most of the FeS proteins. The 51-kDa subunit binds NADH and contains one flavin mononucleotide and one [4Fe4S] cluster as the redox cofactors. The 24-kDa subunit is probably involved in NADH binding and possesses one [2Fe2S] cluster. These two subunits were isolated from Trichomonas vaginalis hydrogenosomes as a heterodimer (Tvh47 and Tvh24) and their ability to transfer electrons from NADH to [2Fe2S] ferredoxin was demonstrated (Hrdy et al. 2004). In addition to the 51- and 24-kDa subunits, the gene coding for the 75-kDa subunit of complex I was found in the genome of N. ovalis (Boxma et al. 2005). Neither ubiqinone nor rodoquinone has been detected in hydrogenosomes (Dyall et al. 2004b); thus, ferredoxin is likely the principal electron acceptor for the hydrogenosomal NADH dehydrogenase. No other FeS proteins of the promotory part were found in the complete genome of T. vaginalis; however, both T. vaginalis and N. ovalis contain genes coding for some subunits of the hydrophobic part (Boxma et al. 2005; Tachezy, unpublished results). Interestingly, the N. ovalis genome also contains two genes encoding mitochondrial complex II subunits including the FeS protein SDH-ß (Boxma et al. 2005).

Hydrogenase is the canonical enzyme of the hydrogenosomes. Although hydrogenosomes have evolved several times in different eukaryotic lineages, they all contain hydrogenases, which belong to the iron-only [Fe] hydroge-nase class (Horner et al. 2000). These hydrogenases are characterized by the presence of an oxygen-sensitive catalytic site, named the H-cluster. It consists of a [4Fe4S] cluster bridged by a cysteinyl residue to a binuclear [2Fe] center. The H-cluster, which is located in the interior of the proteins, is linked to additional [2Fe2S] and [4Fe4S] clusters, which are required for electron transport from soluble mediators (Vignais et al. 2001). The presence of a phylogenetically distinct hydrogenase of the iron-nickel-selenium class was reported only in hydrogenosomes of the anaerobic fungus Neocallimastix frontalis L2 (Marvin Sikkema et al. 1993); however, the identity of the putative [NiFeSe]-hydrogenase was not confirmed by microsequencing and attempts to provide evidence for the presence of corresponding genes in chytrids were not successful (Voncken et al. 2002). In contrast, [Fe]-hydrogenase was later identified in N. frontalis L2 hydrogenosomes by two independent groups (Davidson et al. 2002; Voncken et al. 2002).

In T. vaginalis, there are at least four types of [Fe] hydrogenases, which possess different arrangements of N-terminal electron-transporting clusters and other functional domains (Fig. 6.2):

1. The 50-kDa [Fe] hydrogenase contains two electron-transporting [4Fe4S] clusters preceding the H-cluster. This "short" [Fe] hydrogenase has also been found in some organisms that do not harbor hydrogenosomes, including the parasitic protists Giardia, Spironucleus (Horner et al. 2000), and Entamoeba (Nixon et al. 2003), and hydrogen photoproducing green algae (Vignais et al. 2001).

2. Genes coding a putative hydrogenase with an additional N-terminal [2Fe2S] cluster of the ferredoxin-type were revealed by sequencing of the T. vaginalis genome.

3. The "long" 64-kDa [Fe] hydrogenase was partially purified from T. vaginalis by Payne et al. (1993) and the corresponding gene was later isolated and analysed by Horner et al. (2000). This metalloenzyme possesses an N-terminal [2Fe2S] cluster followed by three [4Fe4S] clusters. It is

2Fe2S 4Fe4S 4Fe4S 4Fe4S

H cluster

CCCC CCCC CCCC

CCCC

CC

C

CC

¡.vaginalis - 64kDa

CCCC CCCC CCCC

CCCC

CC

C

CC

N.frontalis - 70kDa

CCCC CCCC

CCCC

CC

C

CC

¡.vaginalis- 60kDa

CCCC

CCCC

CC

C

CC

^J T.vaginalis - 50kDa

CCCC

CCCC

CC

C

CC

G.intestina!is - 52kDa

CCCC

CCCC

CC

C

CC

E.histolytica - 51kDa

CCCC

CCCC

CC

C

CC

i E.histolytica - 55kDa

CCCC

CC

C

CC

W T.vaginalis Narf

CRP domain

flavodoxin FAD NAD

CCCC CCCC

CCCC

"wc

C

CC

CCCC 1 vaginalis - 120kDa

CCCC CCCC CCCC

CCCC

CC

C

CC

CCCC CCCC J N.ovalis- 130kDa

Complex I subunit 75kDa (NuoG)

24kDa (NuoE)

CCCC CCCC CCCC

CCCC

CCCC

51kDa(NuoF)

CCCC

Fig. 6.2. [Fe] hydrogenases in protists harboring hydrogenosomes and mitosomes. Putative signals targeting the proteins into the hydrogenosomes are in dark blue. Conserved cysteines (C) involved in coordination of [2Fe2S], [4Fe4S], and the H-cluster are in pink boxes, yellow boxes, and red boxes, respectively. Trichomonas vaginalis fusion hydrogenase contains a carboxyl-terminal diflavin domain with similarities to the NADPH-cytochrome P450 oxidore-ductase (CRP), while the fusion hydrogenase of Nyctotherus ovalis contain at its carboxyl terminus two domains with homology to the 24- and 51-kDa subunits of mitochondrial complex I. W typical tryptophan at the carboxyl terminus of T. vaginalis Narf homologue

Fig. 6.2. [Fe] hydrogenases in protists harboring hydrogenosomes and mitosomes. Putative signals targeting the proteins into the hydrogenosomes are in dark blue. Conserved cysteines (C) involved in coordination of [2Fe2S], [4Fe4S], and the H-cluster are in pink boxes, yellow boxes, and red boxes, respectively. Trichomonas vaginalis fusion hydrogenase contains a carboxyl-terminal diflavin domain with similarities to the NADPH-cytochrome P450 oxidore-ductase (CRP), while the fusion hydrogenase of Nyctotherus ovalis contain at its carboxyl terminus two domains with homology to the 24- and 51-kDa subunits of mitochondrial complex I. W typical tryptophan at the carboxyl terminus of T. vaginalis Narf homologue noteworthy that the same arrangement of FeS clusters is present in the 75-kDa (NuoG) subunit of mitochondrial complex I. The evolutionary relationship between some components of complex I and hydrogenase has been suggested (Finel 1998; Pilkington et al. 1991). 4. Finally, T. vaginalis possesses genes encoding "fused" proteins which consist of an N-terminal long-type [Fe] hydrogenase domain and a C-terminal diflavin domain with similarities to the NADPH-cytochrome P450 oxidoreductase (CPR) domain of pyruvate:NADP oxidoreductase (PNO) of Euglena gracilis and C. parvum (Rotte et al. 2001). The "fused" hydrogenase in T. vaginalis is thus distinct from the hydrogenase found in N. ovalis, which contains a C-terminal domain with homology to the 51-kDa (NuoF) and the 24-kDa (NuoE) subunits of complex I (Akhmanova et al. 1998). It can be inferred that T. vaginalis "fused" hydrogenases may catalyze NAD(P)-dependent formation of hydrogen; however, their physiological role has yet to be established.

[2Fe2S] ferredoxin is a principal electron carrier in trichomonad hydrogeno-somes that is required for both pyruvate-as well as malate-dependent catab-olism. The hydrogenosomal ferredoxin is of mitochondrial (adrenodoxin) type, displaying low redox potential (Yarlett et al. 1986b). In addition to its physiological function, it is a key molecule for reducing activation of metron-idazole and other 5-nitroimidazole chemotherapeutics that are used for treatment of trichomoniasis and other anaerobic infections (Kulda 1999; Upcroft and Upcroft 2001). A single copy gene was believed to code the hydrogenosomal ferredoxin in T. vaginalis (Johnson et al. 1990). Crystallographic studies of recombinant protein corresponding to this gene showed its close structural similarity with andrenodoxin, with the exception of a unique cavity close to the FeS center, which was suggested to be responsible for the high rate of metronidazole activation (Crossnoe et al. 2002). Unexpectedly, genomic knockout of the ferredoxin gene resulted neither in inhibition of hydrogenosomal energy metabolism, nor in decreased sensitivity of the trichomonads to metronidazole (Land et al. 2004). More recently, the analysis of the T. vaginalis genome revealed the presence of at least seven distinct genes encoding [2Fe2S] ferredoxins, which explained the previous observations and suggested that hydrogenosomal electron transport is more complex than previously thought. The genes encoding [2Fe2S] ferredoxins were also identified and characterized in Tritrichomonas foetus (Marczak et al. 1983; Suchan et al. 2003) and P. lanterna (Brul et al. 1994).

In mitosome-harboring organisms such as G. intestinalis and E. histolyt-ica, the pyruvate-dependent ATP synthesis as well as transport of electrons generated during carbohydrate catabolism take place in the cytosol; hence, the FeS proteins PFO and ferredoxin are localized within this cellular compartment (Müller 1988, 2003). The cytosolic ferredoxins of Giardia and Entamoeba are of the bacterial type, containing either two cubane [4Fe4S] clusters or one [4Fe4S] and one [3Fe4S] cluster (Nixon et al. 2002a). A [2Fe2s]

ferredoxin of the adrenodoxin type with an N-terminal targeting sequence was found in Giardia and Cryptosporidium, but not in E. histolytica. These proteins are most likely targeted to mitosomes in order to function as a component of the iron cluster assembly machinery.

Recently, [Fe] hydrogenases have been shown to be expressed in Giardia and Entamoeba (Lloyd and Harris 2002; Nixon et al. 2003). In Giardia, a single gene coding "short"-type hydrogenase was found to be transcribed (Nixon et al. 2003) and hydrogenase activity, as well as hydrogen production by the cell, was detected (Lloyd et al. 2002). E. histolytica possesses two different genes. One encodes a "short"-type hydrogenase, similar to that in trichomonads and Giardia, and the other encodes a putative 55-kDa hydro-genase, which appears to be more related to eubacterial [Fe] hydrogenases. Although a recombinant short-type hydrogenase of E. histolytica displays hydrogenase activity, this activity was not found in Entamoeba cells. The cellular localization of giardial and entamoebal hydrogenases has not yet been clarified. The presence of an N-terminal sequence similar to that required for organellar targeting has been suggested; however, no experimental evidence is available (Nixon et al. 2003).

An unusual and most likely cytosolic FeS enzyme of C. parvum is PNO, which consists of an N-terminal PFO domain followed by a C-terminal CPR (Rotte et al. 2001). Unlike the PNO of E. gracilis, which is targeted into mitochondria, the PNO of Cryptosporidium does not possess an N-terminal targeting extension and its presence in a "relict mitochondrion" is therefore unlikely.

A novel essential FeS protein found in the eukaryotic cytosol, is the ATP-binding cassette (ABC) protein Rlilp from Saccharomyces cerevisiae (Decottignies and Goffeau 1997). Unlike ABC transporters, this soluble protein is not a transporter and instead carries two N-terminal cysteine motifs predicted to coordinate two [4Fe4S] clusters. Although the exact role of these clusters is not known, Rli1p is essential for cell viability and is required for maturation and nuclear export of ribosome subunits and for efficient initiation of translation (Dong et al. 2004; Kispal et al. 2005; Yarunin et al. 2005). Homologues of Rlilp are present in all eukaryotes, including hydrogenosome- and mitosome-bearing protists, and in archaebacteria.

Cytosolic aconitase, also named iron-regulatory protein (IRP-1), plays a pivotal role in iron homeostasis of animal cells; yet, this protein has not been found in hydrogenosome- or mitosome-harboring organisms. Interestingly, unlike other eukaryotes, T. vaginalis possesses genes coding for the hybrid cluster protein (HCP) (originally called "prismane"), which coordinates a cubane [4Fe4S] center and a hybrid cluster comprising both O and S bridges between iron atoms (van den Berg et al. 2000). The protein is probably localized in the trichomonad cytosol (Tachezy, unpublished results). Previously, HCP was found in obligate and facultative anaerobic bacteria, for which its involvement in anaerobic nitrate and/or nitrite respiration was suggested. In eukaryotes, HCP-coding genes have only been identified in the genomes of anaerobic aerotolerant protists (G. intestinalis, Spironucleus barkhanus and E. histolytica), providing an example of lateral gene transfer from prokaryotes (Andersson et al. 2003; Han et al. 2004).

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