Engineering of fluorescent reporters into redox domains to monitor electron transfers.
The rate of electron transfer through multicomponent redox systems is often monitored by following the absorbance change due to the oxidation of the upstream pyridine nucleotide electron donor (NADPH or NADH) that initiates the process. Such coupled assay systems are powerful, but because of problems regarding the rate-limiting step, they sometimes limit the kinetic information that can be obtained about individual components. For peroxiredoxins, such assays have led to widespread underestimates of their catalytic power. We show here how this problem can be addressed by a protein engineering strategy inspired by some bacterial and eukaryotic thioredoxins for which a significant fluorescence signal is generated during oxidation that provides a highly sensitive tool to directly measure electron transfers into and out of these domains. For the N-terminal domain of AhpF (a flavoprotein disulfide reductase) and Escherichia coli glutaredoxin 1, two cases not having such fluorescence signals, we have successfully added "sensor" tryptophan residues using the positions of tryptophan residues in thioredoxins as a guide. In another thioredoxin-fold redox protein, the bacterial peroxiredoxin AhpC, we used chemical modification to introduce a disulfide-bonded fluorophore. This modified AhpC still serves as an excellent substrate for the upstream AhpF electron donor but now generates a strong fluorescence signal during electron transfer. These tools have fundamentally changed our understanding of the catalytic power of peroxiredoxin systems and should also be widely applicable for improving quantitative assay capabilities in other electron transfer systems.
Parsonage
Derek
D
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA.
Reeves
Stacy A
SA
Karplus
P Andrew
PA
Poole
Leslie B
LB
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D056890:Eukaryota / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D015202:Protein Engineering; D016415:Sequence Alignment; D013880:Thioredoxin-Disulfide Reductase / Q000032:analysis / Q000737:chemistry / Q000235:genetics
D005456:Fluorescent Dyes; D013880:Thioredoxin-Disulfide Reductase