Electron Spin Resonance and Electron Nuclear Double Resonance Studies of Flavoproteins Involved in the Photosynthetic Electron Transport in the Cyanobacterium Anabaena sp. PCC 7119

The flavins of ferredoxin–NADP + reductase (FNR) and flavodoxin from the cyanobacterium Anabaena PCC 7119 were obtained in their semiquinone states at pH 7 by photoreduction of the pure proteins in the presence of EDTA and 5‐deazariboflavin. For FNR, the ESR signal of the FAD semiquinone was centred at g = 2.005 with linewidths 2.0 mT in H 2 O and 1.48 mT in D 2 O. These data are in agreement with those reported for other neutral flavin semiquinones. The linewidths were the same when measured either at X‐band (9.35 GHz) or at S‐band (4 GHz), indicating that line broadening is due to unresolved nuclear hyperfine couplings, caused in part by exchangeable protons. When the substrate, NADP + , was added to the semiquinone form of the protein no changes in the linewidth or shape of the spectra were detected, but a decrease in the ESR signal due to the FNR semiquinone was observed, consistent with the reduction of NADP + to NADPH by reduced FNR and, subsequent displacement of the equilibrium. No changes in the shape or linewidth of the FNR ESR signals were observed when photoreduction of FNR was performed in the presence of either flavodoxin or ferredoxin. Electron nuclear double resonance (ENDOR) spectroscopy of FNR semiquinone from Anabaena PCC 7119 provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 5–9.5 MHz, is attributed to protons on C6 and on 8‐CH 3 of the flavin ring. No change in these hyperfine couplings was detected when the protein was studied in D 2 O, but the coupling A iso attributed to protons on 8‐CH 3 decreased from 8.12 MHz to 7.72 MHz in the presence of NADP + . The decrease in the electron spin density distribution on this part of the flavin ring system was attributed to binding of the substrate, polarising the electron density distribution of the flavin towards the pyrimidine ring. A second group of signals was observed, with hyperfine couplings less than 3 MHz, some of which disappeared when the protein was transferred into D 2 O. Effects of NADP + binding to the protein were also observed in these weak couplings. These signals are attributed to displaced water protons, or to exchangeable protons from amino acid residues on the protein near the flavin‐binding site, involved in substrate stabilization. For Anabaena flavodoxin the ESR signal of the semiquinone was also centred at g = 2.005, with a linewidth of 2.0 mT, decreasing to 1.47 mT in D 2 O, also corresponding to a neutral semiquinone signal. ENDOR of flavodoxin semiquinone from Anabaena PCC 7119 also provides details of the interaction of the flavin radical with protons. Signals, attributed to protons on 8‐CH 3 and C6 of the flavin ring have also been detected, as well as couplings attributed to water protons, or exchangeable protons from amino acid residues of the protein near the flavin‐binding site. The data are in agreement with the three‐dimensional structure reported for flavodoxin, which shows the flavin ring surrounded mainly by hydrophobic residues.