Abstract SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein. Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1–HSP90 interaction by promoting ternary complex formation.
EPR studies of bacterioferritin (BFR), an iron‐storage protein of Escherichia coli [1993, Biochem. J. 292, 47‐56.], have revealed the presence of non‐haem iron (III) (NHI) sites within the protein coat which may be involved in iron uptake and release. When nitric oxide was used as an EPR spin probe of the Fe(II) state of the NHI sites, two distinct mononuclear NHI species were found. Under certain conditions, an iron dimer was also observed. The reaction of phosphate with NHI species has been investigated. Results point to a function for this anion in core nucleation.
Ferritin proteins function to detoxify, solubilize and store cellular iron by directing the synthesis of a ferric oxyhydroxide mineral solubilized within the protein's central cavity. Here, through the application of X-ray crystallographic and kinetic methods, we report significant new insight into the mechanism of mineralization in a bacterioferritin (BFR). The structures of nonheme iron-free and di-Fe2+ forms of BFR showed that the intrasubunit catalytic center, known as the ferroxidase center, is preformed, ready to accept Fe2+ ions with little or no reorganization. Oxidation of the di-Fe2+ center resulted in a di-Fe3+ center, with bridging electron density consistent with a μ-oxo or hydro bridged species. The μ-oxo bridged di-Fe3+ center appears to be stable, and there is no evidence that Fe3+species are transferred into the core from the ferroxidase center. Most significantly, the data also revealed a novel Fe2+ binding site on the inner surface of the protein, lying ∼10 Å directly below the ferroxidase center, coordinated by only two residues, His46 and Asp50. Kinetic studies of variants containing substitutions of these residues showed that the site is functionally important. In combination, the data support a model in which the ferroxidase center functions as a true catalytic cofactor, rather than as a pore for the transfer of iron into the central cavity, as found for eukaryotic ferritins. The inner surface iron site appears to be important for the transfer of electrons, derived from Fe2+ oxidation in the cavity, to the ferroxidase center. Bacterioferritin may represent an evolutionary link between ferritins and class II di-iron proteins not involved in iron metabolism.
The bacterioferritin (BFR) of Escherichia coli consists of 24 identical subunits, each containing a dinuclear metal-binding site consisting of two histidines and four carboxylic acid residues. Earlier studies showed that the characterization of iron binding to BFR could be aided by EPR analysis of iron-nitrosyl species resulting from the addition of NO to the protein [Le Brun, Cheesman, Andrews, Harrison, Guest, Moore and Thomson (1993) FEBS Lett. 323, 261-266]. We now report data from gas chromatographic head space analysis combined with EPR spectroscopy to show that NO is not an inert probe: iron(II)-BFR catalyses the reduction of NO to N2O, resulting in oxidation of iron(II) at the dinuclear centre and the subsequent detection of mononuclear iron(III). In the presence of excess reductant (sodium ascorbate), iron(II)-BFR also catalyses the reduction of NO to N2O, giving rise to three mononuclear iron-nitrosyl species which are detectable by EPR. One of these, a dinitrosyl-iron complex of S = 1/2, present at a maximum of one per subunit, is shown by EPR studies of site-directed variants of BFR not to be located at the dinuclear centre. This is consistent with a proposal that the diferric form of the centre is unstable and breaks down to form mononuclear iron species.
Journal Article The structure of cytochrome c and its relation to recent studies of long-range electron transfer Get access Gary J. Pielak, Gary J. Pielak Search for other works by this author on: Oxford Academic PubMed Google Scholar David W. Concar, David W. Concar Search for other works by this author on: Oxford Academic PubMed Google Scholar Geoffrey R. Moore, Geoffrey R. Moore 1School of Chemical Sciences, University of East AngliaNorwich NR4 7TJ, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Robert J. P. Williams Robert J. P. Williams Search for other works by this author on: Oxford Academic PubMed Google Scholar Protein Engineering, Design and Selection, Volume 1, Issue 2, February 1987, Pages 83–88, https://doi.org/10.1093/protein/1.2.83 Published: 01 February 1987
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