title: Non-covalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity.
abstract: Although electrostatic interactions between negatively charged ferredoxin (Fd) and positively charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We investigated interprotein forces for the formation of an electron transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400 mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum ∼40-70 mM NaCl and a reverse bell-shaped dependence of interprotein affinity. Meanwhile, intrinsic SiR activity, as measured in a methyl viologen-dependent assay, exhibited saturation above 100 mM NaCl. Thus, two assays suggested that interprotein interaction is crucial in controlling Fd-dependent SiR activity. Calorimetric analyses showed the monotonic decrease in interprotein affinity on increasing NaCl concentrations, distinguished from a reverse bell-shaped interprotein affinity observed from Fd-dependent SiR activity assay. Furthermore, Fd:SiR complex formation and interprotein affinity were thermodynamically adjusted by both enthalpy and entropy through electrostatic and non-electrostatic interactions. A residue-based NMR investigation on the addition of SiR to15N-labeled Fd at the various NaCl concentrations also demonstrated that a combination of electrostatic and non-electrostatic forces stabilized the complex with similar interfaces and modulated the binding affinity and mode. Our findings elucidate that non-electrostatic forces are also essential for the formation and modulation of the Fd:SiR complex. We suggest that a complex configuration optimized for maximum enzymatic activity near physiological salt conditions is achieved by structural rearrangement through controlled non-covalent interprotein interactions.
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: 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: Cellular Function Imaging Team, Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo 650-0047, Japan Multi-Modal Microstructure Analysis Unit, RIKEN CLST-JEOL Collaboration Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kume
authors.affiliation: Department of Plant Physiology, University of Osnabrueck, 11 Barbara Strasse, Osnabrueck 49069, Germany.
authors.forename: Hanke
authors.initials: H
authors.lastname: Gt
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: School of Molecular and Cellular Biology, University of Leeds, Leeds, U.K.
authors.forename: John E
authors.initials: JE
authors.lastname: Ladbury
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: Structural Epigenetics Laboratory, Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, 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: 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: Yuji
authors.initials: Y
authors.lastname: Goto
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.
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Lee
mesh_terms: D002151:Calorimetry; D002942:Circular Dichroism; D004579:Electron Transport / Q000187:drug effects; D005288:Ferredoxins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D050889:Sulfite Reductase (Ferredoxin) / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D005288:Ferredoxins; D012965:Sodium Chloride; D050889:Sulfite Reductase (Ferredoxin)
keywords: electron transfer complex; enzyme activity; ferredoxin; isothermal titration calorimetry; nuclear magnetic resonance; sulfite reductase