title: Structure-function relationships in Anabaena ferredoxin/ferredoxin:NADP(+) reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography.
abstract: The interaction between reduced Anabaena ferredoxin and oxidized ferredoxin:NADP(+) reductase (FNR), which occurs during photosynthetic electron transfer (ET), has been investigated extensively in the authors' laboratories using transient and steady-state kinetic measurements and X-ray crystallography. The effect of a large number of site-specific mutations in both proteins has been assessed. Many of the mutations had little or no effect on ET kinetics. However, non-conservative mutations at three highly conserved surface sites in ferredoxin (F65, E94 and S47) caused ET rate constants to decrease by four orders of magnitude, and non-conservative mutations at three highly conserved surface sites in FNR (L76, K75 and E301) caused ET rate constants to decrease by factors of 25-150. These residues were deemed to be critical for ET. Similar mutations at several other conserved sites in the two proteins (D67 in Fd; E139, L78, K72, and R16 in FNR) caused smaller but still appreciable effects on ET rate constants. A strong correlation exists between these results and the X-ray crystal structure of an Anabaena ferredoxin/FNR complex. Thus, mutations at sites that are within the protein-protein interface or are directly involved in interprotein contacts generally show the largest kinetic effects. The implications of these results for the ET mechanism are discussed.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, University of Arizona, 1041 E. Lowell Street, Tucson, AZ 85721-0088, USA.
authors.forename: John K
authors.initials: JK
authors.lastname: Hurley
authors.forename: Renaud
authors.initials: R
authors.lastname: Morales
authors.forename: Marta
authors.initials: M
authors.lastname: Martínez-Júlvez
authors.forename: Tammy B
authors.initials: TB
authors.lastname: Brodie
authors.forename: Milagros
authors.initials: M
authors.lastname: Medina
authors.forename: Carlos
authors.initials: C
authors.lastname: Gómez-Moreno
authors.forename: Gordon
authors.initials: G
authors.lastname: Tollin
mesh_terms: D017033:Anabaena / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013054:Spectrophotometry, Atomic; D013329:Structure-Activity Relationship
chemical_list: D005287:Ferredoxin-NADP Reductase