C-terminal residues of ferredoxin-NAD(P) reductase from Chlorobaculum tepidum are responsible for reaction dynamics in the hydride transfer and redox equilibria with NADP/NADPH.

Photosynth Res 2018 Jun 8;136(3):275-290. Epub 2017 Nov 8.

Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, Takaramachi 13-1, Kanazawa, Ishikawa, 920-0934, Japan.

Ferredoxin-NAD(P) reductase ([EC 1.18.1.2], [EC 1.18.1.3]) from Chlorobaculum tepidum (CtFNR) is structurally homologous to the bacterial NADPH-thioredoxin reductase (TrxR), but possesses a unique C-terminal extension relative to TrxR that interacts with the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group. In this study, we introduce truncations to the C-terminal residues to examine their role in the reactions of CtFNR with NADP and NADPH by spectroscopic and kinetic analyses. The truncation of the residues from Tyr326 to Glu360 (the whole C-terminal extension region), from Phe337 to Glu360 (omitting Phe337 on the re-face of the isoalloxazine ring) and from Ser338 to Glu360 (leaving Phe337 intact) resulted in a blue-shift of the flavin absorption bands. The truncations caused a slight increase in the dissociation constant toward NADP and a slight decrease in the Michaelis constant toward NADPH in steady-state assays. Pre-steady-state studies of the redox reaction with NADPH demonstrated that deletions of Tyr326-Glu360 decreased the hydride transfer rate, and the amount of reduced enzyme increased at equilibrium relative to wild-type CtFNR. In contrast, the deletions of Phe337-Glu360 and Ser338-Glu360 resulted in only slight changes in the reaction kinetics and redox equilibrium. These results suggest that the C-terminal region of CtFNR is responsible for the formation and stability of charge-transfer complexes, leading to changes in redox properties and reactivity toward NADP/NADPH.

Download full-text PDF

Source
http://link.springer.com/10.1007/s11120-017-0462-z
Publisher Site
http://dx.doi.org/10.1007/s11120-017-0462-zDOI Listing
June 2018
5 Reads

Publication Analysis

Top Keywords

chlorobaculum tepidum
8
c-terminal extension
8
hydride transfer
8
ferredoxin-nadp reductase
8
isoalloxazine ring
8
c-terminal residues
8
c-terminal
5
truncations caused
4
caused slight
4
slight increase
4
blue-shift flavin
4
absorption bands
4
bands truncations
4
flavin absorption
4
intact blue-shift
4
constant nadp
4
michaelis constant
4
constant nadph
4
nadph steady-state
4
steady-state assays
4

References

(Supplied by CrossRef)

A Aliverti et al.
1999

A Aliverti et al.
Arch Biochem Biophys 2008

CJ Batie et al.
J Biol Chem 1984

CJ Batie et al.
J Biol Chem 1986

DS Berkholz et al.
J Mol Biol 2008

G Blankenhorn et al.
Eur J Biochem 1975

A Bortolotti et al.
Biochim Biophys Acta 2014

EA Ceccarelli et al.
Biochim Biophys Acta 2004

CC Correll et al.
Protein Sci 1993

S Daff et al.
BioChemistry 2004

O Dym et al.
Protein Sci 2001

Similar Publications