title: 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.
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Misaki
authors.initials: M
authors.lastname: Kinoshita
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Ju Yaen
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Cellular Function Imaging Team, Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo 650-0047, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kume
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Sakakibara
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Sugiki
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Chojiro
authors.initials: C
authors.lastname: Kojima
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Genji
authors.initials: G
authors.lastname: Kurisu
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Takahisa
authors.initials: T
authors.lastname: Ikegami
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Hase
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan. Electronic address: a-yoko@protein.osaka-u.ac.jp.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kimata-Ariga
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan. Electronic address: mr0505@protein.osaka-u.ac.jp.
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Lee
keywords: Binding thermodynamics; Driving force plot; Electron transfer; Electrostatic interaction; Enzyme activity; Hydrophobic interaction