Reduction and Methyl Transfer Kinetics of the α Subunit from Acetyl Coenzyme A Synthase

Abstract

RECEIVED DATE (will be automatically inserted after manuscript is accepted)Acetyl-coenzyme A synthase is a bifunctional enzyme found in anaerobic archaea and bacteria that grow autotrophically on simple inorganic compounds such as CO 2 /H 2 or CO. The enzyme from Moorella thermoacetica (ACS) is an α 2 β 2 tetramer containing two unique Ni-Fe-S active sites connected by a molecular tunnel. 1,2 The Ni 1 Fe 4 S 4-5 C-cluster catalyzes the reversible reduction of CO 2 to CO and is located in the β subunit. CO generated at this site migrates through the tunnel to the Acluster, located in α, where it reacts with CoA and a methyl group to generate acetyl-CoA. During catalysis, the two sites are mechanistically coupled.The oxidized form of the A-cluster has a Ni 2+ ion bridged to an [Fe 4 The kinetics of this process has been studied by stopped-flow. 4,5 In this technique, solutions of reactants are rapidly mixed and a reaction-sensitive chromophore is monitored by UV-vis spectroscopy. Reaction [1] can be monitored at 390 nm or 450 nm, wavelengths sensitive to CoFeSP. Fitting the data required a three-step model involving: a) docking of ACS and CH 3 -Co 3+ FeSP; b) methyl transfer, and c) undocking of the two proteins. 5 Apparent overall forward and reverse second-order rate constants corresponding to [1] (k f ~ 12 µM -1 sec -1 ; k r ~ 2 µM -1 sec -1 ) indicate that methyl transfer was reversible (K eq ~ 6) and fast.The α subunit of ACS can be cloned and overexpressed in E. coli. 6 Using CO as a substrate, recombinant α catalyzes the synthesis of acetyl-CoA, but at a rate ~10-times slower than ACS. The catalytic mechanism used by α is undoubtedly similar to that used by ACS, allowing mechanistic investigations unfettered by the β subunit. The goals of this study were to investigate the roles of the Fe 4 S 4 component of the A-cluster and the D-site, and to understand the relationship between the two, if any. For example, this component could be the D-site itself or an electron transfer conduit for reducing it.We wondered whether the methyl group of CH 3 -Co 3+ FeSP also transferred reversibly to isolated α. To evaluate this, α was preincubated in Ti 3+ citrate (to reduce D ox ) and then reacted with CH 3 -Co 3+ FeSP. 7 The data ( Figure 1A) demonstrated that the methyl group transferred, albeit slower than with ACS. The reaction was completed within ~1 sec (the equivalent reaction with ACS completes within ~ 0.2 sec). Nearly identical rates were obtained at 450 nm. The methyl group also transferred in the reverse direction, from CH 3 -α to Co 1+ FeSP ( Figure 1B), again at rates significantly slower than with ACS. Experiments were repeated using various protein concentrations (Table 1). Previously described kinetic data fitting procedures and criteria were employed. 5 Differential equations were generated from models and numerically integrated using a set of candidate rateconstants. Simulations were fitted using the Simulated Annealing algorithm. In contrast to the situation with ACS, methyl group transfer data c...

Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase

“…It is important to design experiments that would identify the one-electron shuttle in the synthesis. This is intriguing, since stopped-flow studies suggest that the most likely candidate, the [Fe 4 S 4 ] component of the A cluster, is not involved in redox reactions during the synthesis (32).…”

“…On the other hand, Barondeau and Lindahl (8) proposed that methylation of ACS is the first step in an ordered mechanism of acetyl-CoA synthesis because the methyl group of the CH 3 -CFeSP could be transferred to CODH⅐ACS (in the absence of CO), forming a methylated enzyme that, when treated with CO and CoA, forms acetyl-CoA. That methylation of CODH⅐ACS or recombinant (ACS HT ) (31,32) by CH 3 -CFeSP occurs faster than acetyl-CoA synthesis also suggests that methylation is the first step in the synthesis (33). Furthermore, CO acts as a substrate inhibitor of acetyl-CoA synthesis (34,35).…”

Synthetic Analogues of the Active Site of the A-Cluster of Acetyl Coenzyme A Synthase/CO Dehydrogenase: Syntheses, Structures, and Reactions with CO

“…Although recent studies have indicated that electron flow to and from the Fe 4 S 4 cluster is too slow compared to the rate of methyl transfer, 46 the proposed mechanisms of ACS activity mostly involve redox changes at the Fe 4 S 4 cluster during key steps of the catalytic cycle. 5,6,47,48 Theoretical studies suggest that the Ni(0) state 49 is only stabilized by either removal of the cluster 15 or by the presence of a neighboring reduced iron-sulfur cluster [Fe 4 S 4 ] 1+ (S = ½).…”

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

“…13 Therefore, it has been postulated that reductive activation begins with the CO-dependent reduction of the CODH C cluster that then transfers an electron to the [Fe 4 S 4 ] 2+ cluster of the CFeSP, which reduces the bound Factor III m to the Co 1+ state. 14,15,80 Reduction activation is enhanced by replacement of the lower axial MBI ligand of Factor III m by a weaker water ligand when the cofactor binds to the CFeSP, generating a fivecoordinate Co 2+ species that resembles Co 2+ Cbi + (see above). This ligand switch has a profound influence in the redox properties of the Co center, increasing the Co 2+/1+ reduction potential by ~120 mV relative to that of the MBI-bound form of the cofactor found in solution (~−620 mV).…”