Sandbox Reserved 496: Difference between revisions

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Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional protein which acts as both an [http://en.wikipedia.org/wiki/Oxidoreductase oxidoreductase] and a [http://en.wikipedia.org/wiki/Transferase transferase] by reducing carbon dioxide to carbon monoxide (or the reverse oxidation of carbon monoxide to carbon dioxide) and then catalyzing the synthesis of [http://en.wikipedia.org/wiki/Acetyl-CoA acetyl-CoA] from carbon monoxide, coenzyme A, and the methyl group of an corrinoid iron-sulfur protein.  This enzyme plays a key role in the Wood-Ljungdahl pathway which is used by anaerobic, [http://en.wikipedia.org/wiki/Autotroph autotrophic] bacteria such as ''Moorella thermoacetica'' (f. ''Clostridium thermoaceticum'') and ''Clostridium ljungdahlii'' for gaseous carbon fixation.  End products of the Wood-Ljungdahl pathway include cell biomass, acids (ex. acetate and butyrate), and alcohols (ex. ethanol and butanol) – all of which derive from acetyl-CoA.  The Wood-Ljungdahl pathway is also the most energetically favorable carbon fixation pathway, but its use is confined to only obligate, anaerobic species <ref name="Berg">PMID:20453874</ref>.  Since the discovery of the Wood-Ljungdahl pathway in the early 1980’s, significant effort has been put into trying to characterize the substrate binding activity of CODH/ACS with the ''M. thermoacetica'' protein used as the model in most case studies <ref name="CN">PMID:19583207</ref>.  However, little progress was made in defining the exact structure of the protein with crystal structures until the 2000’s.  Several reasons why CODH/ACS has received so much attention include the fact that it contains highly disputed metalloclusters, its use of biological organometallic intermediates in reactions, and its contribution to reducing environmental pollutants <ref>PMID: 11841199</ref> <ref>PMID: 11848835</ref>.  

<scene name='Sandbox_Reserved_496/Beta_metalloclusters/1'>Five of the metalloclusters</scene> (<font color='red'>'''two B-'''</font>, <font color='blue'>'''two C-'''</font>, and <font color='green'>'''one D-cluster'''</font>) are located in the β2 portion of the protein with a B- and C-cluster in each β subunit and the sole D-cluster bridging the two subunits.  The B- and D-clusters consist of [Fe4S4] centers that serve to transfer electrons.  As the active site in each β subunit where CODH activity occurs, the <scene name='Sandbox_Reserved_496/Beta_c_cluster/2'>C-clusters are composed of a Fe -[NiFe3S4] cage </scene> with the unique Fe coordinated by a bridging sulfur ion, His283, and Cys317 <ref name="Zn">PMID:12627225</ref>.  The B- and D-clusters are positioned in relation to the α-helical domain of the β subunit, while the C-clusters are aligned through interactions with both Rossmann-like domains <ref name="Xe"/>.  A ligand binding study with the native CODH/ACS structure and with cyanide used as competitive inhibitor for CO showed that H2O/OH- binds the C-cluster at the unique Fe and CO binds the C-cluster at the Ni ion <ref name="CN"/>.

The remaining two metalloclusters (both A-clusters) are located in the two α subunits of the CODH/ACS protein.  As the active site of ACS activity in each α subunit, the A-clusters are composed of a [Fe4S4] center bridged to a binuclear site.  There is some debate regarding the metal ions present in the binuclear site; either <scene name='Sandbox_Reserved_496/Alpha_a_cluster/1'>both A-clusters are [Fe4S4]-Cu-Ni</scene> <ref name="Cu"/> or one A-cluster is <scene name='Sandbox_Reserved_496/A_cluster_ni_ni/1'>[Fe4S4]-Ni-Ni</scene>  while the other is <scene name='Sandbox_Reserved_496/A_cluster_zn_ni/1'>[Fe4S4]-Zn-Ni</scene>  <ref name="Zn"/>.  The [Fe4S4] center is coordinated by Cys506, Cys509, Cys518, and Cys528 with Cys509 also forming the bridge between the [Fe4S4] and the [http://en.wikipedia.org/wiki/Proximal#Proximal_and_distal proximal] metal ion.  Furthermore, the distal metal ion is coordinated by Cys595, Gly596, and Cys597with Cys595 and Cys597 also connecting the distal and the proximal metal ions.  Only the [Fe4S4]-Ni-Ni  A-cluster is in an open conformation, the other two site types with Cu or Zn as the proximal ion are in closed form.       

X-ray crystallography methods were used to determine the structure of CODH/ACS from ''M. thermoacetica''.  Crystals were formed by means of sitting drop vapor diffusion.  Both molecular replacement (using the CODH structure of ''R. rubrum'' <ref name="Cu"/> and ''C. hydrogenoformans'' <ref name="Zn"/>) and multiple-wavelength anomalous dispersion (MAD) methods were used to solve the structure’s diffraction pattern.  Resolution of the structures at the highest resolution shell ranges from 2.5Å to 1.9Å.  

 

C-cluster (CODH activity): CO2 + 2H⁺ + 2e^-  ↔  CO + H2O

A-cluster (ACS activity): CH3-Co(III)-CFeSP + CO + HSCoA  ↔  CH3-CO-SCoA +  Co(I)-CFeSP + H⁺

Mechanism 2 proposes that CO binds to the proximal Ni ion after exiting the tunnel.  The A-cluster then changes from the closed to the open conformation which closes the hydrophobic CO tunnel and provides a site for methyl group binding on the proximal Ni as it transitions from Ni(0) to Ni(II).  Next, the bound CO inserts into the Ni-CH3 bond to produce an acetyl intermediate.  Lastly, deprotonated CoA-S- attacks the carbonyl carbon of the acetyl group to produce acetyl-CoA and the proximal Ni is reduced back to Ni(0) <ref name="Zn"/>.  Note that this mechanism differs from the former by binding both CO and CH3 to the proximal metal ion and also the A-cluster stays open between methylation and CoA acetylation.

In the proposed catalytic action of the C-cluster, CO first binds the Ni ion followed by deprotonation of the Fe-bound water to yield a reactive hydroxide which promotes nucleophilic attack of CO by the hydroxide.  Next, the Ni-COOH intermediate is deprotonated to Ni-COO^- which allows release of CO2 as the C-cluster is reduced to a new redox state.  Then, electrons are shuttled between the B- and D-clusters to reoxidize the C-cluster to its original redox state <ref name="CN"/>.  This catalytic cycle is of course operated in reverse to reduce CO2 to CO and allow subsequent production of acetyl-CoA .

Since CODH/ACS consumes CO2 it plays a direct part in the reduction of greenhouse gases by converting CO2 into an intermediate which can transformed into more desirable products such as alcohols and acids.  A novel application in which CODH/ACS plays a role is the production of bioethanol from renewable natural resources such wood, grasses, and agricultural residues (ex. corn stover).  In this process, the biomass undergoes [http://en.wikipedia.org/wiki/Gasification gasification] to yield CO, CO2, and H2.  The gases are subsequently fed to bacterial reactor systems containing organisms which utilize the Wood-Ljungdahl pathway and thus produce ethanol.  The main goal is to produce a renewable energy source that may be used as an alternative to petroleum.  However, at this time, the flux of carbon through the Wood-Ljungdahl pathway does not tend to favor ethanol production so much work remains to be done in this area.  On the other hand, the Wood-Ljungdahl pathway does produce significant levels of acetic acid, and to this extent, the pathway is considered a biological equivalent to the [http://en.wikipedia.org/wiki/Monsanto_process Monsanto process] for industrial acetic acid production <ref name="Berg"/>.  Roughly 10% of the total biological acetic acid production is attributable to anaerobic, autotrophic bacteria which possess CODH/ACS enzymes.