1qof: Difference between revisions

Revision as of 15:41, 21 February 2008

FERREDOXIN MUTATION Q70K

OverviewOverview

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.

About this StructureAbout this Structure

1QOF is a Single protein structure of sequence from Anabaena sp. with and as ligands. Full crystallographic information is available from OCA.

ReferenceReference

Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants., Hurley JK, Weber-Main AM, Stankovich MT, Benning MM, Thoden JB, Vanhooke JL, Holden HM, Chae YK, Xia B, Cheng H, Markley JL, Martinez-Julvez M, Gomez-Moreno C, Schmeits JL, Tollin G, Biochemistry. 1997 Sep 16;36(37):11100-17. PMID:9287153

Page seeded by OCA on Thu Feb 21 14:41:56 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 1: / Line 1: @@
-[[Image:1qof.gif|left|200px]]<br /><applet load="1qof" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1qof.gif|left|200px]]<br /><applet load="1qof" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1qof, resolution 1.8&Aring;" />
 '''FERREDOXIN MUTATION Q70K'''<br />
 ==Overview==
-A combination of structural, thermodynamic, and transient kinetic data on, wild-type and mutant Anabaena vegetative cell ferredoxins has been used to, investigate the nature of the protein-protein interactions leading to, electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+, reductase (FNR). We have determined the reduction potentials of wild-type, vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants, at seven surface residues of vegetative ferredoxin, as well as the one-, and two-electron reduction potentials of FNR, both alone and in complexes, with wild-type and three mutant ferredoxins. X-ray crystallographic, structure determinations have been carried out for six of the ferredoxin, mutants. None of the mutants showed significant structural changes in the, immediate vicinity of the [2Fe-2S] cluster, despite large decreases in, electron-transfer reactivity (for E94K and S47A) and sizable increases in, reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the, relatively small changes in Calpha backbone atom positions which were, observed in these mutants do not correlate with the kinetic and, thermodynamic properties. In sharp contrast to the S47A mutant, S47T, retains electron-transfer activity, and its reduction potential is 100 mV, more negative than that of the S47A mutant, implicating the importance of, the hydrogen bond which exists between the side chain hydroxyl group of, S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations, that alter both reduction potential and electron-transfer reactivity are, E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have, reduction potentials and electron-transfer reactivity that are similar to, those of wild-type ferredoxin. In electrostatic complexes with recombinant, FNR, three of the kinetically impaired ferredoxin mutants, as did, wild-type ferredoxin, induced large (approximately 40 mV) positive shifts, in the reduction potential of the flavoprotein, thereby making electron, transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues, (S47, F65, and E94) on the surface of ferredoxin have large parallel, effects on both the reduction potential and the electron-transfer, reactivity of the [2Fe-2S] cluster and that the reduction potential, changes are not the principal factor governing electron-transfer, reactivity. Rather, the kinetic properties are most likely controlled by, the specific orientations of the proteins within the transient, electron-transfer complex.
+A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.
 ==About this Structure==
-QOF is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Anabaena_sp. Anabaena sp.] with SO4 and FES as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1QOF OCA].
+QOF is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Anabaena_sp. Anabaena sp.] with <scene name='pdbligand=SO4:'>SO4</scene> and <scene name='pdbligand=FES:'>FES</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1QOF OCA].
 ==Reference==
@@ Line 13: / Line 13: @@
 [[Category: Anabaena sp.]]
 [[Category: Single protein]]
-[[Category: Benning, M.M.]]
+[[Category: Benning, M M.]]
-[[Category: Holden, H.M.]]
+[[Category: Holden, H M.]]
 [[Category: FES]]
 [[Category: SO4]]
@@ Line 21: / Line 21: @@
 [[Category: iron-sulfur]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 00:50:44 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:41:56 2008''