Characterization of two ferredoxin-dependent sulfite reductases having different substrate specificity in the red alga Cyanidioschyzon merolae.

Assimilatory sulfite reductase (SiR) and nitrite reductase (NiR), which are important determinants in biomass productivity, are homologous enzymes that catalyze the reduction of sulfite to sulfide and nitrite to ammonium, respectively. They have a siroheme and a [4Fe-4S] cluster as prosthetic groups in common. The red alga Cyanidioschyzon merolae encodes two SiR-like enzymes, CmSiRA and CmSiRB, which are likely products of recent gene duplication, but no homologues of NiR.

Energetic basis on interactions between ferredoxin and ferredoxin NADP reductase at varying physiological conditions.

In spite of a number of studies to characterize ferredoxin (Fd):ferredoxin NADP reductase (FNR) interactions at limited conditions, detailed energetic investigation on how these proteins interact under near physiological conditions and its linkage to FNR activity are still lacking. We herein performed systematic Fd:FNR binding thermodynamics using isothermal titration calorimetry (ITC) at distinct pH (6.0 and 8.

Non-covalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity.

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.

Biochemical and Biophysical Methods to Examine the Effects of Site-Directed Mutagenesis on Enzymatic Activities and Interprotein Interactions.

Mutations in proteins often affect interactions with partner molecules, sequentially changing their activities and functions. In order to examine mutagenic effects, we herein describe practical and detailed protocols for enzymatic activity assays using ferredoxin (Fd)-NADP reductase (FNR) and sulfite reductase (SiR), which are electron-transferring enzymes for the Calvin cycle and sulfur assimilation in various organisms, respectively. Methods for isothermal titration calorimetry and nuclear magnetic resonance spectroscopy, which are very useful thermodynamically and mechanically for investigating the effects of mutations on intermolecular interactions, are also described with practical examples of the Fd-FNR binding system.

Structural and mutational studies of an electron transfer complex of maize sulfite reductase and ferredoxin.

The structure of the complex of maize sulfite reductase (SiR) and ferredoxin (Fd) has been determined by X-ray crystallography. Co-crystals of the two proteins prepared under different conditions were subjected to the diffraction analysis and three possible structures of the complex were solved. Although topological relationship of SiR and Fd varied in each of the structures, two characteristics common to all structures were found in the pattern of protein-protein interactions and positional arrangements of redox centres; (i) a few negative residues of Fd contact with a narrow area of SiR with positive electrostatic surface potential and (ii) [2Fe-2S] cluster of Fd and [4Fe-4S] cluster of SiR are in a close proximity with the shortest distance around 12 Å.

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

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.

NMR characterization of the interaction of the endonuclease domain of MutL with divalent metal ions and ATP.

MutL is a multi-domain protein comprising an N-terminal ATPase domain (NTD) and C-terminal dimerization domain (CTD), connected with flexible linker regions, that plays a key role in DNA mismatch repair. To expand understanding of the regulation mechanism underlying MutL endonuclease activity, our NMR-based study investigated interactions between the CTD of MutL, derived from the hyperthermophilic bacterium Aquifex aeolicus (aqMutL-CTD), and putative binding molecules. Chemical shift perturbation analysis with the model structure of aqMutL-CTD and circular dichroism results revealed that tight Zn(2+) binding increased thermal stability without changing secondary structures to function at high temperatures.