Kinetics of methyl group transfer between the Ni-Fe-S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the stopped-flow method at 390 nm. Rates of the reaction CH(3)-Co(3+)FeSP + ACS(red) <==> Co(1+)FeSP + CH(3)-ACS(ox) in both forward and reverse directions were determined using various protein and reductant concentrations. Ti(3+)citrate, dithionite, and CO were used to reductively activate ACS (forming ACS(red)). The simplest mechanism that adequately fit the data involved formation of a [CH(3)-Co(3+)FeSP]:[ACS(red)] complex, methyl group transfer (forming [Co(1+)FeSP]:[CH(3)-ACS(ox)]), product dissociation (forming Co(1+)FeSP + CH(3)-ACS(ox)), and CO binding yielding a nonproductive enzyme state (ACS(red) + CO <==> ACS(red)-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACS(red) in accordance with K(D) = 180 +/- 90 microM. Fits were unimproved when >1 CO was assumed to bind. Ti(3+)citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH(3)-ACS(ox). This redox site is oxidized by 2e(-) when the methyl cation is transferred from CH(3)-Co(3+)FeSP to ACS(red), and is reduced during the reverse reaction. Best-fit K(D) values for pre- and post-methyl-transfer complexes were 0.12 +/- 0.06 and 0.3 +/- 0.2 microM, respectively. Intracomplex methyl group transfer was reversible with K(eq) = 2.3 +/- 0.9 (k(f)/k(r) = 6.9 s(-1)/3.0 s(-1)). The nucleophilicity of the [Ni(2+)D(red)] unit appears comparable to that of Co(1+) cobalamins. Reduction of the D-site may cause the Ni(2+) of the A-cluster to behave like the Ni of an organometallic Ni(0) complex.

Steady-state initial rates of acetyl-CoA synthesis (upsilon/[E(tot)]) catalyzed by acetyl-CoA synthase from Clostridium thermoaceticum (ACS) were determined at various partial pressures of CO and CO2. When [CO] was varied from 0 to 100 microM in a balance of Ar, rates increased sharply from 0.3 to 100 min(-1). At [CO] > 100 microM, rates declined sharply and eventually stabilized at 10 min(-1) at 980 microM CO. Equivalent experiments carried out in CO2 revealed similar inhibitory behavior and residual activity under saturating [CO]. Plots of upsilon/[E(tot)] vs [CO2] at different fixed inhibitory [CO] revealed that Vmax/[E(tot)] (kcat) decreased with increasing [CO]. Plots of upsilon/[E(tot)] vs [CO2] at different fixed noninhibitory [CO] showed that Vmax/[E(tot)] was insensitive to changes in [CO]. Of eleven candidate mechanisms, the simplest one that fit the data best had the following key features: (a) either CO or CO2 (at a designated reductant level and pH) activate the enzyme (E' + CO right arrow over left arrow E, E' + CO2/2e-/2H+ right arrow over left arrow E); (b) CO and CO2 are both substrates that compete for the same enzyme form (E + CO right arrow over left arrow ECO, E + CO2/2e-/2H+ right arrow over left arrow ECO, and ECO --> E + P); (c) between 3 and 5 molecules of CO bind cooperatively to an enzyme form different from that to which CO2 and substrate CO bind (nCO + ECO right arrow over left arrow (CO)nECO), and this inhibits catalysis; and (d) the residual activity arises from either the (CO)nECO state or a heterogeneous form of the enzyme. Implications of these results, focusing on the roles of CO and CO2 in catalysis, are discussed.

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...

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Robust fitting of core level photoemission spectra is often central to reliable interpretation of X-ray photoelectron spectroscopy (XPS) data. One key element is employment of the correct line shape function for each spectral component. In this study, we consider this topic, focusing on XPS data from atomic adsorbates, namely O and S, on Fe(110). The potential of employing density functional theory (DFT) for generating adsorbate projected electronic density of states (PDOS) to support line shape selection is explored. O 1s core level XPS spectra, acquired from various ordered overlayers of chemisorbed O, all display an equivalent asymmetric line shape. Previous work suggests that this asymmetry is a result of finite O PDOS in the vicinity of the Fermi level, allowing O 1s photoexcitation to induce a weighted continuum of final states through electron-hole pair excitation. This origin is corroborated by O DFT-PDOS generated for an optimised 5 layer Fe(110)(2x2)-O slab.