Flavoproteins involved in photosynthetic electron transport in the cyanobacterium Anabaena sp PCC 7119. Electron spin-echo envelope modulation spectroscopic studies

Abstract

The pulsed EPR technique of electron spin echo modulation (ESEEM) has been utilized to examine the flavoproteins ferredoxin-NADP⁺ reductase (FNR) and flavodoxin from the cyanobacterium Anabaena PCC 7119 in their semiquinone states. Fourier transforms of the three-pulse ESEEM showed the presence of prominent nuclear modulation frequencies around 3.08 MHz for both flavoproteins. Moreover, another prominent component was also observed at 3.47 MHz in FNR. Broader components were observed for the flavosemiquinones in the 4.7 MHz region. These modulations are consistent with the presence of at least one ¹⁴N magnetically coupled to the paramagnet and can be interpreted as arising from nitrogens at positions 1 and/or 3 of the flavin ring system. In FNR, distortion of the ESEEM spectrum was observed in the presence of NADP⁺, while 2′,5′-ADP, an NADP⁺ analogue which lacks the nicotinamide ring, did not show major differences from the spectrum of the native enzyme. This indicates a specific interaction of the flavosemiquinone with the nicotinamide ring, which produces a change in the electron distribution of the flavin ring system. Exchange in deuteriated buffer gave rise to deuteron modulations in ferredoxin-NADP⁺ reductase semiquinone, indicating the presence of exchangeable protons or water molecules in the flavin ring neighbourhood. The results, together with previous ENDOR studies, provide structural information which is complementary to the reported X-ray crystallographic structures.