Structure–function relationships in Anabaena ferredoxin/ferredoxin:NADP+ reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography

AbstractThe 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