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 to N-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.

Controlling Redox Enzyme Orientation at Planar Electrodes

“…This electrostatic interaction allows both partners to come to a close distance which then induces fast intermolecular ET. Various other striking examples of electrostatic-driven protein-protein interactions for efficient ET are documented in the literature: interaction between cytochrome c oxidase and cytochrome c [45], sulfite reductase and ferredoxin [46], cytochrome P450 and putidaredoxin [47], and NADH-cytochrome b 5 reductase and Fe(III)-cytochrome b 5 [48]. Also, the Cu T1 in LACs is inserted in a hydrophobic pocket which allows favorable interaction with its natural organic substrates [49].…”

Functional Interplay between P5 and PDI/ERp72 to Drive Protein Folding

“…The binding isotherms after the subtraction of dilution heat and the baseline correction were fitted to one set of site binding model incorporated in the MicroCal Origin 7.0 software. ([ 47 ]): where Q is the total heat constant and n indicates the number of P5 Cys55/58/190/193Ala mutant molecules, which bind to one of the PDIs. ∆ H is the change in enthalpy for the intermolecular interactions.…”

Structural basis of the correct subunit assembly, aggregation, and intracellular degradation of nylon hydrolase

“…T m and Δ H were determined by regression analysis using nonlinear least-squares fitting of data to a sigmoidal equation under the assumption of a two-state transition between the folded and heat-denatured states 37 . We performed thermodynamic analyses, although the thermal unfolding of proteins was not confirmed.…”