Abstract
The cytochrome b6f complex, for which a crystal structure has been obtained at 2.5 Å resolution (Hasan and Cramer, Structure, 2014) has a central role in the regulation of oxygenic photosynthetic electron transport. The photosytem I peripheral electron transfer proteins ferredoxin (Fd) and ferredoxin NADP+ reductase (FNR) participate in the pathway of NADP+ reduction. Cyclic electron transfer pathway provides an alternate source of electron transfer-mediated membrane energization for ATP production. The present study focuses on the physical interaction of FNR with the cytochrome b6f complex, with relevance to the participation of FNR in a cyclic pathway. The presence of FNR in purified cytochrome b6f complex has been documented and found to be present in sub-stoichiometric levels (Zhang et al. JBC, 2001). It is known that linear electron transfer does not require direct interaction of the cytochrome complex with FNR. In the present study, isothermal calorimetry (ITC) was used to detect and characterize the interaction of FNR with the dimeric cytochrome b6f complex, derived from a plant (spinach) source, in detergent solution. The interaction is relatively weak, with thermodynamic parameters: Kd, 48 μM; ΔH, −23.3 kJ/mol; ΔS, 3.3 J/mol K, at pH 6.5, and ionic strength of 170 mM. It is of interest that such interactions were not detected at pH 8.0, implying a significant pH dependence. Far-UV circular dichroism (CD) analysis of the structure stability of FNR was employed to analyze the pH dependence of the interactions and the effect of detergent. This analysis reveals the decrease in structure stability of FNR and less efficient interaction with the b6f complex in the presence of the detergent UDM. Study supported by U.S. Department of Energy (DOE DE-SC0018238).
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