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Institutes/ Institute of life sciences research and technologies (iRTSV)/ Unités / Chimie et Biologie des Métaux (CBM)/ Biocatalysis/ Formation of binuclear iron centers and Coenzyme Q biosynthesis

Formation of binuclear iron centers and Coenzyme Q biosynthesis


Assembly of binuclear iron centers and Coenzyme Q biosynthesis in the model eukaryote Saccharomyces cerevisiae
Leader
 
Dr. Fabien Pierrel
 iRTSV/LCBM
CEA Grenoble
17 rue des Martyrs
38 054 Grenoble cedex 09
Phone: (33) 4 38 78 91 10
Fax: (33) 4 38 78 91 24
Coenzyme Q biosynthesis
 
Coenzyme Q (ubiquinone) is a lipophilic organic molecule composed of a substituted benzoquinone and a polyprenyl chain containing 6 units in Saccharomyces cerevisiae (Q6) and 10 in humans (Q10). Q has a well known role as an electron carrier in the mitochondrial respiratory chain and also functions as a membrane soluble antioxidant. Primary Q10 deficiency has now been linked to mutations in six genes of Q biosynthesis and results in severe pathologies. The biosynthesis of Q is mitochondrial and requires at least eleven proteins in yeast (Coq1p-9p, Yah1 and Arh1) (Figure 1). 4-hydroxybenzoate (4-HB) is the long-known aromatic precursor of the benzoquinone ring of Q. Despite intensive research efforts and the biological importance of Q, some biosynthetic steps are still uncharacterized. By using a combination of genetics and biochemistry we are trying to better understand the biosynthesis of Q.

We have recently shown that the mitochondrial ferredoxin Yah1p and the ferredoxin reductase Arh1p are required for Q6 biosynthesis at the C5-hydroxylation step (Figure 1) (Pierrel et al., 2010). We have also demonstrated that para-aminobenzoic acid (pABA) is a precursor of Q6 in yeast (Pierrel et al., 2010). This finding implies that when pABA is used as a precursor, an additional NH2-to-OH conversion in Q6 biosynthesis takes place and we develop genetic approaches to characterize this reaction.



Figure 1: Coenzyme Q biosynthetic pathway in S. cerevisiae.
para-hydroxybenzoate (4-HB) is prenylated by Coq2 and then a total of 3 hydroxylation, 3 methylation and 1 decarboxylation yield Q. We have discovered that para-aminobenzoate (pABA, in blue) is also prenylated by Coq2 and converted into Q. The enzymes that convert prenyl-hydroxybenzoate and prenyl-pABA into Q are likely the same, only an additional deamination-hydroxylatio reaction must occur in the case of prenyl-pABA.

We have proven that Coq6, a proposed flavin-dependent monooxygenase is involved exclusively in one of the three hydroxylation reactions of Q biosynthesis: the C5-hydroxylation (Ozeir et al. 2011). The Q6 biosynthetic defect of mutants deficient for the C5-hydroxylation can be bypassed in S. cerevisiae by adding C5-hydroxylated analogues of 4-HB like vanillic acid or 3,4-dihydroxybenzoic acid (Figure 2). This result opens new perspectives in the treatment of Q deficiencies which, to date, are based on coenzyme Q supplementation. We are now working on the hypothesis that Yah1 and Arh1 could be the in vivo source of electrons to reduce the flavin cofactor in Coq6.



Figure 2: Coq6 is required for the C5-hydroxylation of Q6 biosynthesis but Coq6 deficiency can be bypassed by using analogues of 4-HB.
The pathway leading to Q6 biosynthesis in WT S. cerevisiae cells is shown (above dashed line) with Coq6 implicated together with Yah1 and Arh1 in the C5-hydroxylation, whereas the C1-hydroxylation is catalyzed by an unidentified protein (?). In mutant cells (below dashed line) in which the C5-hydroxylation does not take place (crossed arrow), the C1-decarboxylation (dashed arrow) and the C1-hydroxylation proceed efficiently, leading to the accumulation of 3-hexaprenyl-4-hydroxyphenol (4-HP) when 4-HB is prenylated, or the accumulation of 3-hexaprenyl-4-aminophenol (4-AP) when pABA is prenylated (blue). 3,4-diHB and VA contain an additional C5-hydroxyl (green) or C5-methoxyl (green) and enter the Q6 biosynthetic pathway after prenylation by Coq2, leading to restoration of Q6 biosynthesis in cells deficient for the C5-hydroxylation reaction.
Diiron centers assembly
 
Iron and copper are redox metals essential to many cellular processes. Intracellular transport and delivery of copper to target proteins uses well-characterized metallochaperones. However, for iron, intracellular transport and mobilization remains obscure. Iron is used as a cofactor for metalloproteins as Fe-S clusters, heme, bi or mononuclear iron centers. Biosynthesis of Fe-S clusters and heme is quite well understood but little is known regarding the assembly of binuclear iron centers. Binuclear iron centers are found in essential enzymes in eucaryotes. In yeast, the small subunit of ribonucleotide reductase (Rnr2), the C-4 methyl sterol oxidase (Erg25 involved in ergosterol biosynthesis) and Coq7 (participates in Coenzyme Q biosynthesis) all contain a diiron center. We are working to define the assembly process of binuclear iron centers in vivo in the yeast Saccharomyces cerevisiae by studying Rnr2 and Coq7. The main question to be solved is whether the assembly process is spontaneous or requires the assistance of proteins (e.i. metallochaperone).

The demethoxyubiquinone hydroxylase Coq7:

Coq7 hydroxylase activity depends on its diiron center and is required for the mitochondrial biosynthesis of coenzyme Q, the molecule which shuttles electrons form respiratory complexes I/II to III. Coq7 in vivo activity is measured by quantifying in cells extracts, the levels of the substrate demethoxyubiquinone (DMQ6) and of the final product coenzyme Q6 (Figure 1). These 2 molecules are separated by HPLC and quantified by electrochemical detection (Figure 3).



Figure 3: Quantification of coenzyme Q6 and DMQ6 by electrochemical detection coupled to HPLC.
Cellular lipids were extracted from cells, CoQ4 was added as an internal standard and lipid extracts were injected on a C18 column. Cells depleted for Grx3 and Grx4 show reduced CoQ6 and increased DMQ6 suggesting an inhibition of Coq7.

In collaboration with Roland Lill's group (Marburg, Germany), we have recently shown that two cytosolic glutaredoxins (Grx3 and Grx4) are important for Coq7 in vivo activity (Mühlenhoff et al., 2010). Grx3 and Grx4 are able to bind iron in vivo and different phenotypes of yeast cells depleted for Grx3 and Grx4 are consistent with a role of these 2 proteins in cellular iron delivery.

The small subunit of ribonucleotide reductase (Rnr2):

Hydroxyurea (HU) inhibits ribonucleotide reductase by quenching the tyrosinyl radical in Rnr2 and by dissociating the diiron center present in this protein. Most of the genes required for iron import and cellular distribution in yeast are under the control of 2 transcriptional activators Aft1 and Aft2. A strain in which these transcriptional activators have been deleted is sensitive to HU (Figure 4). We propose that this strain is deficient for the metallation of the diiron center of Rnr2, thus causing sensitivity to HU. We have used an unbiased genetic approach to identify proteins that may help assembly of the diiron center of Rnr2.



Figure 4: Serial dilutions of a WT strain, a Δaft1Δaft2 strain and of the different mutants of the Δaft1Δaft2 strain that were isolated after random mutagenesis. Cells were spotted on YPD agar medium containing or not 150mM hydroxyurea (HU). The Δaft1Δaft2 strain is sensitive to HU whereas the mutants are resistant.

We have isolated mutants of the Δaft1Δaft2 strain which are resistant to HU (Figure 4). These mutants show an overall improvement in iron-dependent phenotypes implying that the mutation does not impact specifically Rnr2 metallation but rather more generally iron metabolism. The identification of the mutations is underway.
 
References
 

Ozeir M, Mühlenhoff U, Webert H, Lill R, Fontecave M and Pierrel F
Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
Chemistry & Biology, 2011, 18(9): 1134–1142

Mühlenhoff U, Molik S, Godoy JR, Uzarska MA, Richter N, Seubert A, Zhang Y, Stubbe J, Pierrel F, Herrero E, Lillig CH and Lill R
Cytosolic monothiol glutaredoxins function in intracellular iron sensing and trafficking via their bound iron-sulfur cluster.
Cell Metabolism, 2010, 12(4): 373-385

Pierrel F, Hamelin O, Douki T, Kieffer-Jaquinod S, Mühlenhoff U, Ozeir M, Lill R and Fontecave M
Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.
Chemistry & Biology, 2010, 17(5): 449-459 - pdf