AcsF Catalyzes the ATP-dependent Insertion of Nickel into the Ni,Ni-[4Fe4S] Cluster of Acetyl-CoA Synthase

Abstract

Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO, CoA, and a methyl-cation to form acetyl-CoA at a unique Ni,Ni-[4Fe4S] cluster (the A-cluster). However, it was unknown which proteins support the assembly of the A-cluster. We analyzed the product of a gene from the cluster containing the ACS gene, cooC2 from Carboxydothermus hydrogenoformans, named AcsF Ch , and showed that it acts as a maturation factor of ACS. AcsF Ch and inactive ACS form a stable 2:1 complex that binds two nickel ions with higher affinity than the individual components. The nickel-bound ACS-AcsF Ch complex remains inactive until MgATP is added, thereby converting inactive to active ACS. AcsF Ch is a MinD-type ATPase and belongs to the CooC protein family, which can be divided into homologous subgroups. We propose that proteins of one subgroup are responsible for assembling the Ni,Ni-[4Fe4S] cluster of ACS, whereas proteins of a second subgroup mature the [Ni4Fe4S] cluster of carbon monoxide dehydrogenases.The cellular maturation of metalloenzymes, previously considered a spontaneous process in vivo, typically depends on a machinery of uptake, storage, processing, and delivery factors (1). How metalloenzymes mature has been investigated for some systems, revealing surprisingly complex maturation pathways (2-8). Enzymes containing nickel, although still relatively small in number, play critical roles in archaea, bacteria, and eukarya, through which they impact the global hydrogen (Ni,Fe-hydrogenase), nitrogen (urease), and carbon (acetylCoA synthase, carbon monoxide dehydrogenase, and methylCoM reductase) cycles (9). For most of these enzymes, we now have a good understanding of how nickel is incorporated into the active site (8, 10).The nickel-enzymes acetyl-CoA synthase (ACS) 2 and carbon monoxide dehydrogenase (CODH) are found in a variety of anaerobic microbes, including bacterial sulfate reducers, acetogens, and hydrogenogens, as well as archaeal methanogens and sulfate reducers, where they act as the prime CO 2 and CO converter (11)(12)(13)(14). ACS and CODH can be found as independent monofunctional enzymes in Carboxydothermus hydrogenoformans (15) but are typically found in other microorganisms as protein complexes: in acetogens ACS and CODH form a bifunctional (␣␤) 2 complex, whereas in methanogens they are part of a large (␣␤␥␦⑀) 8 multienzyme complex (11,13,15,16). CODHs catalyze the reversible reduction of CO 2 to CO at the C-cluster, in which a single nickel ion, embedded within a 3Fe-4S scaffold with an additional iron in exo, binds and activates CO and CO 2 for turnover (17)(18)(19). ACS catalyzes the reversible condensation of CO, CoA, and a methyl-cation donated by the methylated corrinoid iron-sulfur protein to form acetyl-CoA (13). ACS depends on a Ni,Ni-[4Fe4S] cluster (also called A-cluster) for activity, in which the two nickel ions have distinct coordinations: the nickel ion distal to the [4Fe4S] cluster (Ni d ) is coordinated by two amide nitrogen atoms and two cysteine thiolates within a Cys-X-Cys ...

Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO 2 and Oxidation of CO by Clostridium acetobutylicum

“…6). Many CODH loci code for a nickel-inserting accessory protein, which is believed to facilitate the insertion of nickel into the CODH active site (40). Here, expression of the nickel-inserting accessory protein CooC from C. carboxidivorans did increase activity, but only in the case of no nickel supplementation.…”

“…Recently, Gregg et al showed the necessity of a nickel accessory protein, AcsF, to facilitate the insertion of nickel into the active site in Carboxydothermus hydrogenoformans (40). Even though we tested our system with additional expression of the AcsF accessory protein, the in vivo carbon exchange reaction could not be detected.…”

“…As stated above, in all studies examining the ACS activity, highly purified amounts were needed to achieve a carbon exchange reaction between CO and the carbonylcarbon from acetyl-CoA (18,19,40,50). The in vitro ACS activity based on this carbon exchange reaction is typically much slower than the in vivo reaction that supports the growth of acetogens.…”

“…Since the active site of the ACS, the A-cluster, also requires a special Ni-containing center, we set out to investigate the necessity of a Ni accessory protein, AcsF, found in the WLP gene cluster (Ccar_18805). Recently, the Ni insertion accessory protein AcsF from Carboxydothermus hydrogenoformans was shown to be necessary for the ACS active site to fully develop (40). The C. acetobutylicum strain expressing the ACS and the AcsF from C. carboxidivorans did not increase the labeling in CO either.…”

Something special about CO‐dependent CO2 fixation

“…the aerobic and anaerobic CODH enzymes have different subunit structures, different cofactors and are not related at the amino acid sequence or structural level []. Prokaryotes that use aerobic CODH use CO as a source of electrons in energy metabolism, are typically aerobes or facultative aerobes and tend to transfer the electrons from CO to high potential acceptors such as O 2 or acceptors derived from it such as nitrate (NO 3 − ) .…”