Acetyl-coa Synthetase Center Of Metal Structure, Function And Molecular Mechanism Research

Acetyl-coenzyme A synthase (ACS)/carbon monoxide dehydrogenase (CODH) is a bifunctional metalloenzyme found in anaerobic archaea Morrella thermoacetica. It is a α2β2tetramer with a310kD molecular weight, containing seven metal clusters (two A-cluster, two B-cluster, two C-cluster and one D-cluster) connected by a133A molecular tunnel network. The active site A-cluster locates in the a subunit and catalyzes the synthesis of acetyl-coenzyme A (Eq.1): CH3-Co3+FeSP+CO+CoA(?) CH3-C (O)-CoA+Co1+FeSP+H+(Eq.l) The B, D, and C-cluster locate in the β subunit, in which catalyzes the reversible reduction of CO2to CO by the C-cluster (Eq.2): CO2+2e-+2H+(?)CO+H2O (Eq.2)CODH in microorganisms, in combine with plants’photosynthesis, plays a critical role in global carbon cycle. It is estimated that CODH activity accounts for the annual removal-108tons of CO from the environment. As a key substance in material and metabolism, acetyl-coenzyme A plays a central role in energy metabolism in vivo. Anaerobic microorganisms, such as acetogenic and methanogenic bacteria, catalyze the synthesis of acetyl-coenzyme A using CO/CO2as carbon sources and H? as energy sources, respectively. It is estimated that～1011tons of acetate and～109tons of methane are produced globally through this process every year. In the last thirty years, ACS/CODH has become a research focus by biochemical and inorganic scientists, due to complexity and diversity for its metal centers as well as importance for its catalytic reactions.The active site A-cluster, which is located in a the subunit of ACS, consists of an [Fe4S4] cluster coordinated through a bridging cysteinate to a dinuclear nickel sub-component. The A-cluster should be converted to an activated Ared-act state through reduction by two external electrons before every catalytic cycle. In the last ten years, great progress concerning the structure, function and kinetic mechanism of metalloenzyme ACS/CODH has been made, and several state forms of the A-cluster have been identified or proposed to understand the reductive activation of the A-cluster (Ared-act).However, the roles of the sub-components of the active site of the A-cluster and the mechanisms of their interactions remain elusive. Thus, we have carried out intensive and systematic researches for the issue in chapter2and chapter Firstly, to probe the role of bridging C509in A-cluster, four single-substitution mutants (C509A, C509V, C509H and C509S) and one triple-substitution mutant (C509A/S511A/H516A) named△bridge were designed and purified in a glove box under anaerobic atmosphere. The relevant properties of these variants were evaluated in detail by biochemical/biophysical approaches, such as U V/Vis spectra and EPR, etc. The kinetics of the variants’[Fe4S4]2+cubane reduction and methylation were measured by stopped-flow experiments. Our results reveal that the bridging thiolate of C509, which could be partly substituted by imidazole of histidine, is crucial for the methylation activity of ACS. In addition, we proposed that the C509could mediate an interaction between the [Fe4S4] cubane and the [NipNid] sub-site, as well as influence the redox potential of both sub-components.Previously, stopped-flow kinetics of [Fe4S4]+cubane reduction and methylation of the A-cluster monitored by us revealed that the reduction rate of cubane is100-fold slower than that of the methylation. This result contradicts and disproves the mechanistic role for [Fe4S4] as an electron transfer redox intermediary between the [NipNid] and external redox agents. Thus, the mechanistic role of the [Fe4S4] cubane in the A-cluster remains intangible to date. In chapter3, we adopted two strategies to structurally modify the A-cluster by genetics. One strategy is to truncate the gene segments encoded [Fe4S4] cubane, yielding a15kD protein named α15with the C-terminal136residues of ACS. The other is to prepare a triple-substitution mutant called△cubane, in which the three cysteine residues (cys506, cys518and cys528) that coordinate to the iron in the cubane were replaced by alanines, such that the cubane could not form. The crystal structure of Ni-containing α15was crystallized and refined to2.35A, and its Ni-K edge XAFS spectrum were measured as well. The results of these experiments showed that α15has a novel di-nickel center distinct from the one in wild-type ACS. Both α15and△cubane showed no methyl transfer activity and lost the NiFeC EPR signals, indicating that the [Fe4S4] cubane is essential for the catalytic activity of ACS. Next, we treated the Ni-containing α15with benzenethiol, an organic molecule with electron-delocalization ring, to obtain a benzenethiol-bound variant. Unexpectedly, this variant exhibited a slow but detectable level of methyl group transfer activity, indicating that benzenethiol has a "rescue" effect on the methylation activity instead of [Fe4S4] cubane. These results combined with previous studies, have led us to propose that the {R-X-Nip}(R:[Fe4S4], bezenthiol; X:thiolate of Cys509or other bridging residues) might form a "hyper conjugation system", and that the electron delocalization effect could stabilize the activated low valence Nip state. The [Fe4S4] cubane might function as a modulator of redox properties of the Nip, but not as an electron-transfer redox intermediary between the Nip and external redox agents. In summary, the three sub-components of the A-cluster form a co-operative unit to conduct the function of ACS, and each one is indispensible.Metalloproteins account for near half of all proteins in nature, and their metal-binding sites are always responsible for catalyzing numerous important biological processes. Metalloproteins are suitable for modification and functional design due to the efficiency and diversity of their metal cofactors. An ultimate test is to use our knowledge to design new metalloproteins that reproduce the structures and functions of native metalloproteins. In chapter4, we have converted the ACS truncated protein α15into a nickel superoxide dismutase (Ni-SOD) mimic protein through functional design and protein engineering.ACS and Ni-SOD are two known Ni redox metalloenzymes catalyzing distinct biological reactions. Initially, comparing the metal centers in these two enzymes reveal that the distal nickel site (Nid) of ACS and reduced Ni-SOD display similar square-planar NiⅡN2S2coordination environments. The Nid of ACS has been suggested to be redox inactive remaining NiⅡ state, while the Ni-SOD accesses both the NiⅡ and NiⅢ during its catalytic cycles. The lack of axial ligand in Nid of ACS in contrast to His1in oxidant Ni-SOD may cause the huge differences in their physiological functions. In chapter3, we have reported the crystal structure of a truncated ACS protein α15containing only the Nid segments of ACS. However, this variant likewise lacks an axial ligand and shows no SOD activity in either the presence or absence of imidazole. To introduce an axial ligand in Nid site, we firstly prepared two single-substitution mutants (S594H and F598H) based on α15.The SOD activity measurements indicated that both variants obtained obvious SOD activity, corresponding to2850U for F598H and340U for S594H, respectively. Their EPR results in presence of KO2revealed a low-spin five coordination signal of Ni3+, clearly proving that our design of introducing an axial ligand is successful. In addition, the Ni bind affinity of the variants was measured by ITC and glycine competition titration.; the Trp fluorescence emission and ANS fluorescence experiments suggest a Ni-induced structure rearrangements exist in the variants; the cyclic voltammetry results showed that the redox potential of Ni centers in the active variants were all in the ranges of the reaction catalyzed by SOD enzymes. Secondly, to better mimic the secondary coordination sphere of the metal center in Ni-SOD, in particular for H-bond supplied by the conserved residue Tyr9, we designed four double-substitution mutants (S594Y/F598H, S594E/F598H and S594H/F598Y) to introduce a putative H-bond. Furthermore, we used MD and DFT methods to monitor the structures of metal centers in the variants as well as the transition states in the catalytic cycles.In conclusion, we proposed an out-sphere mechanism exists in the active mimic SOD proteins in this study. The crystallographic studies of the mutated proteins are still under way in our lab to better understand their catalytic mechanism, as well as to guide our design in future.There is no efficient method to obtain recombinant CODH by E.coli due to its high insolubility. In the last chapter, we tried several prokaryotic expression plasmids to express and purify recombinant CODH from thermophilic bacteria Moorella thermoacetica. Finally, we have successfully obtained soluble CODH by two methods:(1) co-expression of ACS and CODH;(2) co-expression of the binding domain of ACS to CODH and CODH. In addition, we also tried to co-express another plasmid, which expressed a series of proteins to help assemble the iron-sulfur clusters in CODH. These results lay a solid basis for further studies on the structure-function relationship of CODH as well as the interaction of its metal clusters.