Structure-function relationships in Anabaena ferredoxin/ferredoxin:NADP (+) reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography
Hurley, J.K.; Morales, R.; Martínez-Júlvez, M.; Brodie, T.B.; Medina, M.; Gómez-Moreno, C.; Tollin, G.
Biochimica et Biophysica Acta 1554(1-2): 5-21
2002
ISSN/ISBN: 0006-3002 PMID: 12034466 DOI: 10.1016/s0005-2728(02)00188-3
Accession: 011411546
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.