Physicochemical nature of interfaces controlling ferredoxin NADP+ reductase activity through its interprotein interactions with ferredoxin

Abstract

Although acidic residues of ferredoxin (Fd) are known to be essential for activities of various Fd-dependent enzymes, including ferredoxin NADP(+) reductase (FNR) and sulfite reductase (SiR), through electrostatic interactions with basic residues of partner enzymes, non-electrostatic contributions such as hydrophobic forces remain largely unknown. We herein demonstrated that intermolecular hydrophobic and charge-charge interactions between Fd and enzymes were both critical for enzymatic activity. Systematic site-directed mutagenesis, which altered physicochemical properties of residues on the interfaces of Fd for FNR /SiR, revealed various changes in activities of both enzymes. The replacement of serine 43 of Fd to a hydrophobic residue (S43W) and charged residue (S43D) increased and decreased FNR activity, respectively, while S43W showed significantly lower SiR activity without affecting SiR activity by S43D, suggesting that hydrophobic and electrostatic interprotein forces affected FNR activity. Enzyme kinetics revealed that changes in FNR activity by mutating Fd correlated with Km, but not with kcat or activation energy, indicating that interprotein interactions determined FNR activity. Calorimetry-based binding thermodynamics between Fd and FNR showed different binding modes of FNR to wild-type, S43W, or S43D, which were controlled by enthalpy and entropy, as shown by the driving force plot. Residue-based NMR spectroscopy of (15)N FNR with Fds also revealed distinct binding modes of each complex based on different directions of NMR peak shifts with similar overall chemical shift differences. We proposed that subtle adjustments in both hydrophobic and electrostatic forces were critical for enzymatic activity, and these results may be applicable to protein-based electron transfer systems.

NADP(H) allosterically regulates the interaction between ferredoxin and ferredoxin‐NADP+ reductase

“…While the positive ΔH bind values display energetically unfavorable endothermic binding reaction, negative values of ΔG bind and ÀTΔS bind ( Table 2) indicate spontaneous Fd : FNR complex formation driven by entropy gain (positive ΔS bind ). These parameters of wild-type FNR in the absence of NADP + were very similar to those of our previous analysis under the same condition [18] and similar to other analyses of the same proteins under 25 mM sodium phosphate pH 6.0/50 mM sodium perchlorate at 300 K [23] and of Anabaena Fd and FNR under 50 mM Tris/HCl, pH 8.0 [11]. The resulting dissociation constant (K d ) increased by addition of NADP + into the titration reaction, although the extent of the increase was smaller than that of K m value for Fd upon addition of NADPH ( Table 1).…”

“…The titration of Fd to wild-type FNR showed a series of heat peaks indicating complex formation with heat uptake (Fig. 5), as reported previously [18]. While the positive ΔH bind values display energetically unfavorable endothermic binding reaction, negative values of ΔG bind and ÀTΔS bind ( Table 2) indicate spontaneous Fd : FNR complex formation driven by entropy gain (positive ΔS bind ).…”

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…2b), which ensures fast electron transfer in such weak complexes (Crowley and Ubbink 2003). These interactions require a fine-tuned balance between hydrophobic and electrostatic contributions (Kinoshita et al 2015).…”

Structure of cyanobacterial photosystem I complexed with Cytochrome c6 and Ferredoxin at 1.97 Å resolution

“…Binding of Fd to its partner protein (in this case, PSI) appears to be entirely driven by entropy. Attractive electrostatic interactions, polar interactions, and Van der Waals’ force-driven interactions might contribute to a negative ΔH (ΔH<0), but detachment of water molecules bound to interfaces for complexation of Fd and PSI will cost thermodynamically to give a positive value for the net ΔH (ΔH>0), as also observed in the Fd-FNR complex formation 57,60 . Nonetheless, conformational disorder accompanying the displacement of water molecules from the interface can make complex formation energetically favorable due to the associated increase in entropy.…”

“…To obtain thermodynamic insights into complex formation of Fd with PSI including hydrating water molecules, ITC measurements were performed. Based on analysis of the thermodynamic parameters, the formation of the Fd-PSI complex is akin to that of the Fd-FNR 57 and the Fd:GmHO 58 type (ΔH>0 and ΔS>0) but not to that of the Fd:SiR type 59 (ΔH<0 and ΔS>0). Binding of Fd to its partner protein (in this case, PSI) appears to be entirely driven by entropy.…”