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Kinetic interactions of beta-lactam antibiotics such as penicillin-G and cefotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneumoniae and a penicillin-resistant PBP2x (PBP2x(R)) from a resistant clinical isolate (CS109) of the bacterium have been extensively characterized using electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicillin-G has been demonstrated, and the dissociation constant, K(d) of 0.9 mm, and the acylation rate constant, k(2) of 180 s(-1), have been determined for the first time. The millimolar range K(d) implies that the beta-lactam fits to the active site pocket of the penicillin-sensitive PBP rather poorly, whereas the extremely fast k(2) value indicates that this step contributes most of the binding affinity of the beta-lactam. The values of K(d) (4 mm) and k(2) (0.56 s(-1)) were also determined for PBP2x(R). The combined value of k(2)/K(d), known as overall binding efficiency, for PBP2x(R) (137 m(-1) s(-1)) was over 1000-fold slower than that for PBP2x (200,000 m(-1) s(-1)), indicating that a major part is played by the acylation steps in penicillin resistance. Most of the decreased binding efficiency of PBP2x(R) comes from the decreased ( approximately 300-fold) k(2). Kinetic studies of cefotaxime acylation of the two PBP2x proteins confirmed all of the above findings. Deacylation rate constants (k(3)) for the third step of the interactions were determined to be 8 x 10(-6) s(-1) for penicilloyl-PBP2x and 5.7 x 10(-4) s(-1) for penicilloyl-PBP2x(R), corresponding to over 70-fold increase of the deacylation rate for the resistant PBP2x(R). Similarly, over 80-fold enhancement of the deacylation rate was found for cefotaxime-PBP2x(R) complex (k(3) = 3 x 10(-4) s(-1)) as compared with that of cefotaxime-PBP2x complex (3.5 x 10(-6) s(-1)). This is the first time that such a significant increase of k(3) values was found for a beta-lactam-resistant penicillin-binding protein. These data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2x(R) resistance to beta-lactams.

Methanosarcina thermophila contains a multienzyme complex called the carbon-monoxide dehydrogenase complex, which has been resolved into a nickel/iron-sulfur and a corrinoid/iron-sulfur component. This complex plays a central role in acetoclastic methanogenesis. The Ni/Fe-S component catalyzes CO oxidation and has been proposed to be involved in cleavage of acetyl-CoA into its methyl, carbonyl, and CoA moieties. In the work reported here, three metal centers in the Ni/Fe-S component were characterized by electron paramagnetic resonance (EPR) spectroscopy and spectroelectrochemistry and pre-steady state kinetics. Center A contains nickel and iron and forms an EPR active adduct with CO, which is called the NiFeC species. The EPR spectrum of the NiFeC species has g values of 2.059, 2.051, and 2.029 and is observable at temperatures as high as 150 K. This signal had previously been observed only in the carbon-monoxide dehydrogenase complex of M. thermophila and the acetyl-CoA synthase from acetate-producing bacteria. Incubation of the CO-reduced Ni/Fe-S component with acetyl-CoA resulted in an increase in intensity of the NiFeC signal, which supports a role for the component in the cleavage of acetyl-CoA. Generation of the NiFeC EPR signal occurs with a rate constant of 0.4 s-1, a result that demonstrates the kinetic competence of this species in the acetyl-CoA cleavage reaction but rules it out as the site of oxidation of CO to CO2. Center B is likely to be a [4Fe-4S]2+/1+ center with g values of 2.04, 1.93, and 1.89 (gav = 1.95) and a standard reduction potential (E'0) of -444 mV. At potentials less than -500 mV, another EPR signal develops that appears to originate from another state of Center B. Center C is a fast relaxing center with g values of 2.02, 1.88, and 1.71 (gav = 1.87) and an E'0 of -154 mV.

The corrinoid/iron-sulfur protein (C/Fe-SP) from Clostridium thermoaceticum acts as a methyl group carrier in the anaerobic acetyl-CoA pathway of CO and CO2 fixation. Consisting of a small (approximately 33 kDa) and a large (approximately 55 kDa) subunit, the C/Fe-SP contains 1 mol of cobalt in a corrinoid cofactor and 1 mol of [4Fe-4S]2+/1+ cluster/mol of alpha beta dimer. Cobalt is the site of methylation, and the [4Fe-4S] center appears to serve an electron transfer function. The genes encoding both subunits have been cloned previously and are located within a gene cluster that includes other genes required for CO2 fixation by anaerobic bacteria. When the genes encoding the C/Fe-SP were expressed in Escherichia coli, the protein was found to be inactive. We report the amino acid sequences of the large and small subunits of the C/Fe-SP based on the DNA sequences of the cloned genes. The [4Fe-4S] cluster was found to be located in the large subunit. Although the primary structural lattice for cobamide binding resides in the small subunit, both subunits are required for formation of a stable cobamide-binding protein. Based on sequence comparisons with other [4Fe-4S]-containing proteins, 3 of the 4 cysteine residues that serve as ligands to the iron sites in the cluster have been located. The two subunits were independently overexpressed in E. coli to a level of 30-50% of cell protein; however, the resulting protein was inactive, lacked stoichiometric amounts of Fe-S cluster, and lacked cobamide. By combining the recombinant subunits, unfolding them with urea, and refolding in the presence of cobamide, iron, and inorganic sulfide, the resulting C/Fe-SP was found to contain stoichiometric amounts of cobamide and [4Fe-4S] cluster and had spectroscopic and enzymatic properties similar to those of the native protein. We expect that the methods developed here may be used for heterologous overexpression and reconstitution of other complex metalloenzymes. The C/Fe-SP was found to utilize with equal efficiency either vitamin B12 or the natural cofactor 5-methoxybenzimidazolylcobamide as a methyl carrier.

The multienzyme carbon monoxide dehydrogenase complex from Methanosarcina thermophila contains at least two protein components: a CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and a cobalt-containing corrinoid/iron-sulfur component (Co/Fe-S). The CO dehydrogenase complex has been shown to synthesize acetyl-CoA from CoA, CH3I, and CO as well as to cleave acetyl-CoA into its methyl, carbonyl, and CoA components as the first step in the catabolism of acetyl-CoA to methane and CO2. Presumed to serve as an acceptor of the methyl group of acetyl-CoA en route to methane, the Co/Fe-S component contains iron, acid-labile sulfur, and a corrinoid cofactor (factor III) that is the site of methylation. Using EPR spectroscopy and spectroelectrochemistry, we characterized the cobalt and Fe-S centers of the Co/Fe-S component. The redox and EPR properties of the metal centers in the isolated Co/Fe-S component are similar to those of the Co/Fe-S component in the CO dehydrogenase enzyme complex, a result that indicates that any protein-protein interaction between components in the complex has little influence on the properties of the metal centers. The corrinoid is maintained in the base-off state with a formal equilibrium reduction potential (E'o) at pH 7.8 of -486 mV for the Co2+/1+ couple that facilitates reduction of the Co2+ state by approximately 12 kcal/mol relative to base-on cobamides. The Co/Fe-S component also contains a [4Fe-4S]2+/1+ cluster with an E'o at pH 7.8 of -502 mV, which is nearly isopotential with the Co2+/1+ couple of the cobamide.

The final steps in the synthesis of acetyl-CoA by CO dehydrogenase (CODH) have been studied by following the exchange reaction between CoA and the CoA moiety of acetyl-CoA. This reaction had been studied earlier (Pezacka, E., and Wood, H. G. (1986) J. Biol. Chem. 261, 1609-1615 and Ramer, W. E., Raybuck, S. A., Orme-Johnson, W. H., and Walsh, C. T. (1989) Biochemistry 28, 4675-4680). The CoA/acetyl-CoA exchange activity was determined at various controlled redox potentials and was found to be activated by a one-electron reduction with half-maximum activity occurring at -486 mV. There is approximately 2000-fold stimulation of the exchange by performing the reaction at -575 mV relative to the rate at -80 mV. Binding of CoA to CODH is not sensitive to the redox potential; therefore, the reductive activation affects some step other than association/dissociation of CoA. We propose that a metal center on CODH with a midpoint reduction potential of less than or equal to -486 mV is activated by a one-electron reduction to cleave the carbonyl-sulfur bond and/or bind the acetyl group of acetyl-CoA. Based on a comparison of the redox dependence of this reaction with that for methylation of CODH (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) and CO2 reduction and formation of the Ni-Fe-C EPR signal (Lindahl, P. A., Münck, E., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3873-3879), we propose that the assembly of the acetyl group of acetyl-CoA, i.e. binding the methyl group of the methylated corrinoid/iron-sulfur protein, binding CO, and methyl migration to form the acetyl-CODH intermediate, occur at the novel Ni-Fe3-4-containing site in CODH. CO has two effects on the CoA/acetyl-CoA exchange: it activates the reaction due to its reductive capacity and its acts as a noncompetitive inhibitor. We also discovered that the CoA/acetyl-CoA exchange was inhibited by nitrous oxide via an oxidative mechanism. In the presence of a low-potential electron donor, CODH becomes a nitrous oxide reductase which catalytically converts N2O to N2. This study combined with earlier results (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) establishes that the two-subunit form of CODH is completely active in all reactions known to be catalyzed by CODH.

Many anaerobic bacteria fix CO2 via the Wood pathway of acetyl-CoA synthesis. Carbon monoxide dehydrogenase (CODH), also called acetyl-CoA synthase, accepts the methyl group from the methylated corrinoid/iron-sulfur protein (C/Fe-SP), binds a carbonyl group from CO, CO2, or the carboxyl of pyruvate, and binds coenzyme A. Then CODH catalyzes the synthesis of acetyl-CoA from these enzyme-bound groups. Here, we have characterized the methyl transfer steps involved in acetyl-CoA synthesis. We have studied the reactions leading to methylation of CODH by methyl iodide and shown an absolute requirement of the C/Fe-SP in this reaction. In addition, we have discovered and partly characterized two previously unknown exchange reactions catalyzed by CODH: between the methylated C/Fe-SP and methylated CODH and between methylated CODH and the methyl moiety of acetyl-CoA. We have performed these two exchange reactions, methylation of the C/Fe-SP, and methylation of CODH at controlled potentials. The rates of all these reactions except the exchange between methylated C/Fe-SP and methylated CODH are accelerated (from 1 to 2 orders of magnitude) when run at low potentials. Our results provide strong evidence for a nucleophilic redox-active metal center on CODH as the initial acceptor of the methyl group from the methylated C/Fe-SP. This metal center also is proposed to be involved in the cleavage of acetyl-CoA in the reverse reaction.