pH probes respond to redox changes in cytochrome o

Area of Science:

• Biochemistry
• Membrane Biophysics
• Spectroscopy

Background:

• N-Phenylnaphthylamine (NPN) is a known probe for membrane lipid fluidity.
• Previous work demonstrated NPN fluorescence changes with cytochrome o activity in Escherichia coli.
• pH probes like pyranine and quinacrine are commonly used for measuring vesicle internal pH.

Purpose of the Study:

• To investigate the behavior of pH probes pyranine and quinacrine in response to cytochrome o activity.
• To determine if the observed fluorescence changes are related to pH gradients or probe distribution.
• To elucidate the mechanism behind the fluorescence response of NPN, pyranine, and quinacrine to cytochrome o redox state.

Main Methods:

• Incorporation of purified cytochrome o from Escherichia coli into soybean phospholipid vesicles.
• Monitoring fluorescence intensity changes of NPN, pyranine, and quinacrine.
• Utilizing cyanide to alter the redox state of cytochrome o.
• Testing the effect of uncouplers and varying pH gradients.

Main Results:

• Pyranine and quinacrine exhibit fluorescence changes similar to NPN upon alteration of cytochrome o's oxygen content.
• These fluorescence changes are independent of pH gradients and probe distribution.
• The NPN response is dependent on the presence of phospholipid, while pyranine and quinacrine responses occur even with soluble cytochrome o.
• Cyanide-induced alterations in cytochrome o's redox state correlate with probe fluorescence changes.

Conclusions:

• The fluorescence response of NPN, pyranine, and quinacrine is linked to the redox state of cytochrome o, not pH changes.
• Probes may bind differently or experience environmental changes upon redox state alteration of the cytochrome.
• Caution is advised when using pyranine and quinacrine to measure internal pH in membrane vesicles containing redox proteins like cytochrome o.