Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E^0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the Ni-Ni-Fe₄S₄] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like 2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins.Energy transduction is fundamental for life. Aerobic and anaerobic organisms depend on coupling ATP hydrolysis to movement, activation of metabolites, or peptide bond formation, among others. Several metal-containing enzymes, such as nitrogenase, radical-dependent β,α-dehydratases, the related benzoyl-CoA reductases, and different cobalamin-dependent methyltransferases are able to convert unreactive molecules by acting in a low-potential regime. The highly energetic electrons required for these reactions (1–3) are injected by ATPases that enable the transfer of electrons against the redox potential gradient, driven by the hydrolysis of ATP. Three different types of reductive metallo-ATPase have been described so far.The enzyme nitrogenase is by reducing dinitrogen with six electrons to ammonia at the heart of the global nitrogen cycle (1, 4, 5). Nitrogenase consists of the dinitrogenase, also called MoFe protein for the predominant Mo-containing variant, and the dinitrogenase reductase, called Fe protein (1, 4–6). The Fe protein is a homodimer covalently linked through a 4Fe-4S] cluster bound within the dimer interface. Both monomers are able to bind and hydrolyze ATP in a cleft containing a P loop. For electron transfer (ET) between Fe and MoFe proteins to occur, the reduced Fe protein binds MgATP and forms a complex with the MoFe protein positioning the electron-donating 4Fe-4S] cluster and electron-accepting P cluster within the typical limits for physiological ET (< 15 ?) (1, 4, 7, 8). Hydrolysis of two ATP molecules initiates a one-electron transfer between both partners (9, 10). Conformational changes of the Fe protein induced by ATP hydrolysis are believed to act as switches for the association/dissociation of the Fe:MoFe protein complex and the delivery of electrons (8–11). The Fe protein is bifunctional and also acts as a molybdate/homocitrate insertase during the maturation of nitrogenase (5, 12).Benzoyl CoA reductases and 2-hydroxyacyl CoA dehydratases rely on homologous metallo-ATPases to catalyze the reduction of benzoyl-CoA or the β/α-dehydration of 2-hydroxyacyl-CoA compounds via formation of ketyl radicals (2). The structure of the homodimeric activator of 2-hydroxyglutaryl-CoA dehydratase revealed a 4Fe-4S] cluster covalently linking the two monomers, on a first glance resembling the Fe protein (13). The structure of the activator also showed it to be a member of the ASKHA (acetate and sugar kinases/heat shock protein 70/actin) superfamily. ASKHA proteins catalyze phosphoryl transfers or hydrolysis of ATP in a variety of biological contexts and are distinct from the P loop containing switch-type NTPases to which the Fe protein of nitrogenase belongs (13, 14). The binding of two MgATP molecules to the reduced activator is supposed to induce a conformational change and drives formation of the complex with the dehydratase. ATP hydrolysis likely increases the reducing power of the reduced 4Fe-4S] cluster of the activator, enabling the one-electron transfer to the low-potential 4Fe-4S] cluster of the dehydratase (2, 15). Unlike the 2-hydroxyacyl-CoA dehydratase system, the reduction of benzoyl-CoA is a two-step ET requiring a stoichiometric consumption of ATP (3).Recently, a third class of electron-transferring metallo-ATPases has been discovered (16–18). This enzyme class belongs to the COG3894 protein family and has been termed reductive activases for corrinoid enzymes (RACE) (17). The genome of several anaerobic microorganisms, which encode corrinoid-dependent methyltransferases and enzymes of the reductive acetyl-CoA pathway, also encode for proteins homologous to the two investigated RACE proteins with their characteristic binding motifs for one Fe/S cluster (17, 18). Bacterial RACE proteins typically show 2Fe-2S] cluster-binding-motifs, as in the veratrol O-demethylase system of Acetobacterium dehalogenans (16), whereas in archaea, as in the activator of the methylamine methyltransferase of the methanogenic archaeon Methanosarcina barkeri 4Fe-4S] cluster-binding motifs are more abundant (17, 18).The anaerobic hydrogenogenic bacterium Carboxydothermus hydrogenoformans is able to convert CO₂ into cellular carbon compounds via the reductive acetyl-CoA pathway (also known as the Wood–Ljungdahl pathway) (19–21). The corrinoid/iron-sulfur protein (CoFeSP) connects the methyl and carbonyl branch of this pathway by accepting a methyl group from methyltetrahydrofolate bound to a methyltransferase and donating it to the Ni,Fe-containing acetyl-CoA synthase (22, 23). Three redox states are known for the corrinoid cofactor of CoFeSP: The nucleophilic Co(I) acts as a methyl-acceptor, Co(II) is an oxidized inactive state, and CH₃ - Co(III) acts as the methyl donor of acetyl-CoA synthase (22, 23). The occasional oxidation of Co(I) to Co(II) inactivates CoFeSP, which has to be reactivated by a one-electron reduction (23, 24). The low midpoint potential needed to reduce Co²⁺ to Co¹⁺ (< -504 mV at pH 7.4) (25) can be achieved in vitro using either chemical reducing agents such as Na-dithionite (DT), Ti³⁺ citrate, via photoreduction with deazariboflavin as a catalyst or enzymatically with electrons generated by the oxidation of CO to CO₂ by carbon monoxide dehydrogenase (22, 26). An ATP-dependent reactivation of CoFeSP has not been reported so far.An open reading frame (orf7), situated between the structural genes coding for the CoFeSP subunits CfsA and CfsB of Moorella thermoacetica (27), codes for a member of the COG3894 protein family and contains the putative 2Fe-2S] cluster-binding motif CX₅CX₂CX_nC (17, 18). The genome of C. hydrogenoformans contains a similar arrangement of genes coding for enzymes of the reductive acetyl-CoA pathway as M. thermoacetica, including a homolog of Orf7 (CHY_1224 assigned as COG3894). To test whether an ATP-dependent reductive activator is operative in the reductive acetyl-CoA pathway, we established the heterologous production of the Orf7 homolog and investigated its activity, structure, and selective complex formation with CoFeSP. Furthermore, we reveal its relationship to known ATPases including the activator of 2-hydroxyacyl-CoA dehydratases and compare its strategy to achieve unidirectional electron transport with the other types of ATP-dependent activators/reductases.