Iron-sulfur cluster cysteine-to-serine mutants of Anabaena -2Fe-2S- ferredoxin exhibit unexpected redox properties and are competent in electron transfer to ferredoxin:NADP+ reductase.
The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ reductase (FNR) are reported for site-directed mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anabaena PCC 7120, each of which has a cluster ligating cysteine residue mutated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (wt) Fd. This is a consequence of oxygen replacing one of the ligating cysteine sulfur atoms, thus altering the ligand --> Fe charge transfer transition energies and the chiro-optical properties of the chromophore. Each mutant is able to rapidly accept an electron from deazariboflavin semiquinone (dRfH.) and to transfer an electron from its reduced form to oxidized FNR although all are somewhat less reactive (30-50%) toward FNR and are appreciably less stable in solution than is wt Fd. Whereas the reduction potential of C46S (-381 mV) is not significantly altered from that of wt Fd (-384 mV), the potential of the C49S mutant (-329 mV) is shifted positively by 55 mV, demonstrating that the cluster potential is sensitive to mutations made at the ferric iron in reduced [2Fe-2S] Fds with localized valences. Despite the decrease in thermodynamic driving force for et from C49S to FNR, the et rate constant is similar to that measured for C46S. Thus, the et reactivity of the mutants does not correlate with altered reduction potentials. The et rate constants of the mutants also do not correlate with the apparent binding constants of the intermediate (Fdred:FNRox) complexes or with the ability of the prosthetic group to be reduced by dRfH.. Furthermore, the X-ray crystal structure of the C49S mutant is virtually identical to that of wt Fd. We conclude from these data that cysteine sulfur d-orbitals are not essential for et into or out of the iron atoms of the cluster and that the decreased et reactivity of these Fd mutants toward FNR may be due to small changes in the mutual orientation of the proteins within the intermediate complex and/or alterations in the electronic structure of the [2Fe-2S] cluster.
Hurley
J K
JK
Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
Weber-Main
A M
AM
Hodges
A E
AE
Stankovich
M T
MT
Benning
M M
MM
Holden
H M
HM
Cheng
H
H
Xia
B
B
Markley
J L
JL
Genzor
C
C
Gomez-Moreno
C
C
Hafezi
R
R
Tollin
G
G
D017033:Anabaena / Q000737:chemistry*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D012694:Serine; D005287:Ferredoxin-NADP Reductase; D003545:Cysteine