Kim, Jongsun (1993) Crystallographic structures and functional implications of nitrogenase molybdenum-iron proteins from Azotobacter vinelandii and Clostridium pasteurianum. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-09182007-093920
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Three-dimensional structures of the nitrogenase molybdenum-iron (MoFe-) proteins from Azotobacter vinelandii and Clostridium pasteurianum have been determined by X-ray crystallography. The structure of MoFe-protein from A. vinelandii (Av1) was determined at 2.7[...] by the method of multiple isomorphous replacement (MIR) and noncrystallographic symmetry (NCS) averaging both within and between crystal forms. The Av1 model has been refined to a crystallographic R factor of 19% with good geometry. The root mean square (rms) deviation of bond lengths and bond angles are 0.016[...] and 3.3°, respectively. The structure of MoFe-protein from C. pasteurianum (Cp1) was determined at 3.0[...] by a combination of molecular replacement, single isomorphous replacement (SIR) and NCS averaging both within and between crystal forms. The Cp1 model has been refined to a crystallographic R factor of 18% with good geometry. The rms deviation of and lengths and bond angles are 0.018[...] and 3.9°, respectively.
The MoFe-protein, which is an [alpha]2[beta]2 tetramer with a total molecular weight of ~240kD, contains two types of metal centers: the FeMo-cofactor and the P-cluster pair. The FeMo-cofactor is believed to represent the site of substrate reduction and the P-cluster pair may function in electron transfer between iron (Fe-) protein and the FeMo-cofactor. The FeMo-cofactor contains two clusters of composition 4Fe:3S and lMo:3Fe:3S that are bridged by three non-protein ligands. Two of the bridging ligands are assigned as sulfurs, while the chemical identity of the "Y" ligand is still ambiguous, but it could be sulfur. The Fe-Fe distance between bridged iron sites average ~2.5[...], suggesting that there may be some iron-iron bonding interactions, which could contribute the fourth coordination for the bridging irons. Ignoring the partial iron-iron bonding interactions between bridged irons, six of the seven Fe atoms in the FeMo-cofactor have trigonal coordination geometry, are coordinatively unsaturated, and are potential sites for N2 activation. The N2 binding site in FeMo-cofactor may be relevant to the H2 binding site in the H-cluster of Fe-hydrogenases and the O2 binding site in the Mn-center of PSII. Homocitrate, an essential component of FeMo-cofactor, is coordinated through a hydroxyl and carboxyl oxygen to the molybdenum site. The FeMo-cofactor is attached to the [alpha] subunit through two protein ligands, Cys [alpha]275 and His [alpha]442. The P-cluster pair consists of two 4Fe:4S clusters that are bridged by two cysteine thiol ligands and a disulfide bond between two of the cluster sulfurs. The P-cluster pair is attached at the interface between the [alpha] and [beta] subunits through seven protein ligands: Cys [alpha]62, Cys [alpha]88, Cys [alpha]154, Cys [beta]70, Cys [beta]95, Cys [beta]153, and Ser [beta]188. The structure of the P-cluster pair indicates that the P-cluster pair can act as a two-electron redox group, involving cleavage and reformation of the [mu]3-disulfide bridge coupled to the transfer of electrons into the FeMo-cofactor. This disulfide bond may also provide a site for H2 evolution.
The [alpha] and [beta] subunits in the [alpha]2[beta]2 MoFe-protein tetramer exhibit similar polypeptide folds consisting of three domains of [alpha]/[beta] type with some extra helices. The [alpha] and [beta] subunits of MoFe-protein are related by an approximate two-fold axis which passes through the center of the P-cluster pair, and there are two wide and shallow clefts around the P-cluster pair which may provide the binding site for the dimeric Fe-protein. Docking studies between the Fe-protein and MoFe-protein suggest a possible interaction mode between the two proteins that involves the surface of the MoFe-protein near the approximate two-fold axis passing through the P-cluster pair, and the surface of the Fe-protein near the 4Fe:4S cluster. The overall dimensions of the [alpha]2[beta]2 MoFe-protein teamer are ~70[...] x 80[...] x 110[...]. The two [alpha][beta] subunit pairs are related by a two-fold NCS axis. Even though the [alpha] and [beta] subunits in an [alpha][beta] subunit pair are also approximately related by a two-fold rotation, the MoFe-protein does not exhibit 222 symmetry. The MoFe-protein tetramer interface is stabilized by packing of helices primarily provided by two [beta] subunits, with some contribution from the [alpha] subunit, and further stabilized by divalent cation binding.
The FeMo-cofactor is buried at least 10[...] below the protein surface. No permanent channels between the protein surface and the FeMo-cofactor are present, however, there are two potential clefts which could be utilized for substrate entry/product release and/or H3O+ transport. The protein environment of the FeMo-cofactor indicates that there are multiple potential transfer pathways. The P-cluster pair is also buried about 12[...] below the protein surface and the environment of the P-cluster pair is primarily provided by hydrophobic residues. The edge-edge distance of the FeMo-cofactor to the P-cluster pair is about 14[...]. Four helices are oriented in parallel between the two metal centers and could play a role in electron transfer. In particular, the helices [alpha]63-74 and [alpha]88-92 provide the most direct structural connection between a P-cluster pair and FeMo-cofactor.
The structure of Cp1, including the two types of metal centers associated with the protein, are similar to that of Av1. Unique features of the Cp1 structure arise from the presence of a ~50 residue insertion in the [alpha] subunit and a ~50 residue deletion in the [beta] subunit. As a consequence, the FeMo-cofactor is more buried in Cp1 than in Av1, since the insertion is located on the surface above the FeMo-cofactor. The location of this insertion near the putative Fe-protein binding site provides a structural basis for the observation that the nitrogenase proteins from C. pasteurianum have low activity with complementary nitrogenase proteins isolated from other organisms.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||4 May 1993|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||16 Oct 2007|
|Last Modified:||26 Dec 2012 03:01|
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