Proton conduction through full-length gp91phox requires histidine 115

Area of Science:

• Biochemistry
• Cell Biology
• Enzymology

Background:

• NADPH oxidase is a key enzyme in neutrophils, involved in electron transfer and proton efflux.
• gp91 (phox) is a subunit of NADPH oxidase, proposed to contain essential cofactors and function as the proton conduction pathway.
• Previous studies indicated that mutations in specific histidines within an N-terminal fragment of gp91 (phox) disrupt proton conduction.

Purpose of the Study:

• To investigate the role of specific histidine residues (H111, H115, H119) in proton conduction by the full-length gp91 (phox) subunit.
• To determine if histidine 115 is essential for proton transport in both the N-terminal fragment and the complete gp91 (phox) protein.

Main Methods:

• Generation of stable Chinese Hamster Ovary (CHO) cell lines expressing full-length gp91 (phox) with specific histidine-to-leucine mutations (CHO91H111/115/119 and CHO91H115L).
• Assessment of gp91 (phox) expression and cellular localization in wild-type and mutant cell lines to ensure comparability.
• Measurement of arachidonate-activated proton influx and efflux in the engineered cell lines to quantify proton conduction.

Main Results:

• Mutations of histidines 111, 115, and 119 to leucines, or solely histidine 115 to leucine, resulted in a near-complete loss of proton conduction.
• Proton influx and efflux were significantly reduced in both mutant cell lines compared to wild-type gp91 (phox).
• Expression and localization of gp91 (phox) were comparable across wild-type and mutant cell lines, validating the observed effects on proton conduction.

Conclusions:

• Histidine 115 is essential for proton conduction mediated by both the full-length gp91 (phox) and its N-terminal fragment.
• The findings support a critical role for histidine 115 in the proton transfer mechanism of NADPH oxidase.
• A model for the dual role of histidine 115, potentially involving proton conduction and heme iron coordination, is proposed, drawing parallels with cytochrome c oxidase.