30 research outputs found
Variable-temperature, variable-field magnetic circular dichroism spectroscopic study of NifEN-bound precursor and âFeMocoâ
NifEN plays a key role in the biosynthesis of the ironâmolybdenum cofactor (FeMoco) of nitrogenase. A scaffold protein that hosts the conversion of a FeMoco precursor to a mature cofactor, NifEN can assume three conformations during the process of FeMoco maturation. One, designated ÎnifB NifEN, contains only two permanent [Fe4S4]-like clusters. The second, designated NifENPrecursor, contains the permanent clusters and a precursor form of FeMoco. The third, designated NifENâFeMocoâ, contains the permanent [Fe4S4]-like clusters and a fully complemented, âFeMocoâ-like structure. Here, we report a variable-temperature, variable-field magnetic circular dichroism spectroscopic investigation of the electronic structure of the metal clusters in the three forms of dithionite-reduced NifEN. Our data indicate that the permanent [Fe4S4]-like clusters are structurally and electronically conserved in all three NifEN species and exhibit spectral features of classic [Fe4S4]+ clusters; however, they are present in a mixed spin state with a small contribution from the S > ½ spin state. Our results also suggest that both the precursor and âFeMocoâ have a conserved Fe/S electronic structure that is similar to the electronic structure of FeMoco in the MoFe protein, and that the âFeMocoâ in NifENâFeMocoâ exists, predominantly, in an S = 3/2 spin state with spectral parameters identical to those of FeMoco in the MoFe protein. These observations provide strong support to the outcome of our previous EPR and X-ray absorption spectroscopy/extended X-ray absorption fine structure analysis of the three NifEN species while providing significant new insights into the unique electronic properties of the precursor and âFeMocoâ in NifEN
A combined computational and experimental investigation of the [2Feâ2S] cluster in biotin synthase
Biotin synthase was the first example of what is now regarded as a distinctive enzyme class within the radical S-adenosylmethionine superfamily, the members of which use Fe/S clusters as the sulphur source in radical sulphur insertion reactions. The crystal structure showed that this enzyme contains a [2Feâ2S] cluster with a highly unusual arginine ligand, besides three normal cysteine ligands. However, the crystal structure is at such a low resolution that neither the exact coordination mode nor the role of this exceptional ligand has been elucidated yet, although it has been shown that it is not essential for enzyme activity. We have used quantum refinement of the crystal structure and combined quantum mechanical and molecular mechanical calculations to explore possible coordination modes and their influences on cluster properties. The investigations show that the protonation state of the arginine ligand has little influence on cluster geometry, so even a positively charged guanidinium moiety would be in close proximity to the iron atom. Nevertheless, the crystallised enzyme most probably contains a deprotonated (neutral) arginine coordinating via the NH group. Furthermore, the Fe¡¡¡Fe distance seems to be independent of the coordination mode and is in perfect agreement with distances in other structurally characterised [2Feâ2S] clusters. The exceptionally large Fe¡¡¡Fe distance found in the crystal structure could not be reproduced
Molecular cloning and characterization of ARO7-OSM2, a single yeast gene necessary for chorismate mutase activity and growth in hypertonic medium
Magnetic Circular Dichroism Spectroscopy of Metalloproteins
Metals and metal clusters in proteins typically serve as important structural/functional motifs. Because of this reason, there is a wide range of techniques that specifically probe the structure and energy levels of metals in metalloproteins. One technique, magnetic circular dichroism (MCD) spectroscopy, is the focus of this chapter. MCD spectroscopy monitors the circular dichroism spectrum induced by a magnetic field and is an effective way of obtaining electronic and structural information of paramagnetic metal ions or clusters. The basic methodology of this technique is discussed along with examples of how MCD spectroscopy can be used to elucidate typical metal clusters in proteins. Special emphasis is placed on iron-sulfur (FeS) clusters
Loss of 2 um DNA from Saccharomyces cerevisiae transformed with the chimaeric plasmid pJDB219
Repression of growth-regulated G1 cyclin expression by cyclic AMP in budding yeast.
IF2003=30.97