Cluster, active site

Chen, P., Gorelsky, S.I., Ghosh, S. and Solomon, E.I. (2004) NzO reduction by the i4-sulfide-bridged tetranuclear Cuz cluster active site, Angew. Chem. Int. Ed., 43, 4132-4140. 

Since the first purifications of Fe hydrogenases in the early 1970s a range of different models for the H-cluster active site have been proposed including mononuclear iron and clusters of 2, 3, 4, and 6Fe [56,57,72-77], At least in part this changing stoichiometry reflects improvements in purification, elemental analysis, and spectroscopy. The more recent models propose the H cluster to contain approximately 6 Fe on the basis of elemental analysis [56,57] and a putative 3.3 A Fe-Fe distance indicated by EXAFS spectroscopy [58], The data are indicative at best, because counting Fe in proteins has an uncertainty typically of the order of 10% (i.e., l-2Fe), and because no EXAFS on 6Fe models has been published. 

The low-temperature MCD and absorption titration studies (Figure 10) have determined that azide binds to both the type 2 and type 3 centers with similar binding constants. A series of chemical perturbations and stoichiometry studies have shown that these effects are associated with the same azide. This demonstrates that one N3 bridges between the type 2 and type 3 centers in laccase. These and other results from MCD spectroscopy first defined the presence of a trinuclear copper cluster active site in biology (89). At higher azide concentration, a second azide binds to the trinuclear site in laccase. Messerschmidt et al. have determined from X-ray crystallography that a trinuclear copper cluster site is also present in ascorbate oxidase (87, 92) and have obtained a crystal structure for a two-azide-bound derivative (87). It appears that some differences exist between the two-azide-bound laccase and ascorbate oxidase derivatives, and it will be important to spectroscopically correlate between these sites. 

The cluster model approach assumes that a limited number of atoms can be used to represent the catalyst active site. Ideally, one would like to include a few thousands atoms in the model so that the cluster boundary is sufficiently far from the cluster active site thus ensuring that edge effects are of minor importance and can be neglected. Unfortunately, the computational effort of an ab initio calculation grows quite rapidly with the number of atoms treated quantum mechanically and cluster models used in practice contain 20 to 50 atoms only. It is well possible that with the advent of the N-scaling methods " this number can dramatically increase. Likewise, the use of hybrid methods able to decompose a very large system in two subsets that are then treated at different level of accuracy, or define a quantum mechanical and a classical part, are also very promising. However, its practical implementation for metallic systems remains still indeterminate. 

Spectroscopic stndies indicate a [4Fe S] cluster active site that interacts directly with the substrate disulfide, rather than via an active-site disulfide as in FTR. Moreover, they have provided compelling evidence for a one-electron reduced intermediate, similar to that characterized in FTR, involving a [4Fe-4S] + cluster with both cysteinate and the CoM thiolate bound at a unique Fe site. Hence a similar mechanism to that shown in Figure 8 for FTR is also proposed for the direct [4Fe-4S]-mediated cleavage of the CoM-S-S-CoB heterodisnlfide in methanogenic archaea by HDR. 5... 

Putative pathways have been characterized in C. hydrogenoformans CODH for the respective transit of CO/CO2 and the H2O product through hydrophobic and hydrophiUc tunnels, respectively [87]. The bi-functional CODH/ACS from M. thermoacetica contains several hydrophobic tunnels that connect the two CODH C-cluster active sites to each other and to the ACS active site named the A-cluster [90]. High-pressure, xenon-binding experiments carried out in a CODH/ACS crystal have shown that these tunnels can trap many xenon atoms [91]. In addition, putative proton transfer pathways connecting... 

Fig. 29.20 The structure of the [FeFe]-hydrogenase from the bacterium C. pasteurianum (PDB code 3C8Y). The protein backbone is shown in ribbon representation, and the Fe and S atoms in the [Fe-S] clusters and active site are shown as spheres. The Fl-cluster (active site) is the left-hand cluster highlighted in the diagram. Colour code Fe, green S, yellow C, grey O, red N, blue. See Fig. 29.21 for an enlargement of the active site.

Lee SK, George SD, Antholine WE, Hedman B, Hodgson KO, Solomon EL 2002. Nature of the intermediate formed in the reduction of O2 to H2O at the trinuclear copper cluster active site in native laccase. J Am Chem Soc 124 6180-6193. 

Chen P, Gorelsky SI, Ghosh S, Solomon El. 2004. N2O reduction by the (i4-sulfide-bridged tetranuclear Cu cluster active site. Angew Chem, Int Ed 43 4132-4140. 

Quintanar L, Yoon J, Aznar CP, Palmer AE, Andersson KK, Britt RD, Solomon El. 2005. Spectroscopic and electronic structure studies of the trinuclear Cu cluster active site of the multicopper oxidase laccase nature of its coordination unsaturation. J Am Chem Soc 127 13832-13845. 

Nonrepetitive but well-defined structures of this type form many important features of enzyme active sites. In some cases, a particular arrangement of coil structure providing a specific type of functional site recurs in several functionally related proteins. The peptide loop that binds iron-sulfur clusters in both ferredoxin and high potential iron protein is one example. Another is the central loop portion of the E—F hand structure that binds a calcium ion in several calcium-binding proteins, including calmodulin, carp parvalbumin, troponin C, and the intestinal calcium-binding protein. This loop, shown in Figure 6.26, connects two short a-helices. The calcium ion nestles into the pocket formed by this structure. 

FIGURE 20.7 (a) The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-/ hydrogen from the pro-/ arm of citrate, (b) The active site of aconitase. The iron-sulfur cluster (red) is coordinated by cysteines (yellow) and isocitrate (white). 

The spatial arrangement of the Fe-S clusters in D. gigas NiFe-hydrogenase (see Fig. 1) suggests an active role for the [Fe3S4] ° cluster in mediating electron transfer from the NiFe active site to the... 

These studies of protein-bound heterometallic cubanes have amply demonstrated that the heterometal site is redox active and able to bind small molecules. Although they have yet to be identified as intrinsic components of any protein or enzyme (except as part of the nitrogenase FeMo cofactor cluster (254)), they are clearly attractive candidates for the active sites of redox enzymes. 

Homocitrate is bound to the molybdenum atom by its 2-carboxy and 2-hydroxy groups and projects down from the molybdenum atom of the cofactor toward the P clusters. This end of FeMoco is surrounded by several water molecules (5, 7), which has led to the suggestion that homocitrate might be involved in proton donation to the active site for substrate reduction. In contrast, the cysteine-ligated end of FeMoco is virtually anhydrous. 

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