An electrochemical, kinetic, and spectroscopic characterization of [2Fe-2S] vegetative and heterocyst ferredoxins from Anabaena 7120 with mutations in the cluster binding loop.
Residues within the cluster binding loops of plant-type [2Fe-2S] ferredoxins are highly conserved and serve to structurally stabilize this unique region of the protein. We have investigated the influence of these residues on the thermodynamic reduction potentials and rate constants of electron transfer to ferredoxin:NADP+ reductase (FNR) by characterizing various single and multiple site-specific mutants of both the vegetative (VFd) and the heterocyst (HFd) [2Fe-2S] ferredoxins from Anabaena. Incorporation of residues from one isoform into the polypeptide backbone of the other created hybrid mutants whose reduction potentials either were not significantly altered or were shifted, but did not reconcile the 33-mV potential difference between VFd and HFd. The reduction potential of VFd appears relatively insensitive to mutations in the binding loop, excepting nonconservative variations at position 78 (T78A/I) which resulted in approximately 40- to 50-mV positive shifts compared to wild type. These perturbations may be linked to the role of the T78 side chain in stabilizing an ordered water channel between the iron-sulfur cluster and the surface of the wild-type protein. While no thermodynamic barrier to electron transfer to FNR is created by these potential shifts, the electron-transfer reactivities of mutants T78A/I (as well as T48A which has a wild-type-like potential) are reduced to approximately 55-75% that of wild type. These studies suggest that residues 48 and 78 are involved in the pathway of electron transfer between VFd and FNR and/or that mutations at these positions induce a unique, but unproductive orientation of the two proteins within the protein-protein complex.
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
A M
AM
Weber-Main
J K
JK
Hurley
H
H
Cheng
B
B
Xia
Y K
YK
Chae
J L
JL
Markley
M
M
Martinez-Júlvez
C
C
Gomez-Moreno
M T
MT
Stankovich
G
G
Tollin
D017033:Anabaena / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D002942:Circular Dichroism; D004563:Electrochemistry; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D013816:Thermodynamics
D005288:Ferredoxins; D008670:Metals