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Molecular basis for Rac2 regulation of phagocyte NADPH oxidase

Nature Immunology volume 2, pages 211–215 (2001)Cite this article

Abstract

A Rac GTPase–regulated multiprotein NADPH oxidase is critical for the formation of reactive oxygen species (ROS) in phagocytic leukocytes and other nonphagocytic cells. NADPH oxidase reduces molecular oxygen to form superoxide anion in a two-step process. Electrons are initially transferred from NADPH to cytochrome b–associated FAD, then to cytochrome b heme and finally to molecular oxygen. We show here that Rac is required for both electron-transfer reactions. Mutational and biophysical analysis shows that Rac and p67phox independently regulate cytochrome b to catalyze the transfer of electrons from NADPH to FAD. However, they must interact with each other to induce the subsequent transfer of electrons from FAD to cytochrome b heme and molecular oxygen. This two-step model of regulation by Rac GTPase may provide a means of more effectively controlling the inflammatory responses of phagocytic leukocytes.

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Figure 1: Dependence of NADPH oxidase electron transfer reactions on Rac2 concentration.
Figure 2: NADPH oxidase activity is p47phox independent.
Figure 3: The interaction of Rac2 with p67phox is required for the step 2, but not the Step 1, electron-transfer reaction.
Figure 4: The Rac2 insert domain is required for both step 1 and step 2 electron-transfer reactions.
Figure 5: Physical interaction of Rac2 with cyt b shown by fluorescence of mant-GppNHp-Rac2 complex.

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Acknowledgements

We thank C. DerMardirossian for discussions and advice for cyt b purification; A. Cross for critical comments and technical advice; F. Wientjes for advice on p67phox mutant purification; and B. P. Bohl and J. Lu for technical assistance with protein preparation and molecular biology, respectively. Supported by USPHS grant M01 RR00833 to the General Clinical Research Center at The Scripps Research Institute; NIH grant HL48008 (to G. M. B.) and by a fellowship from the American Heart Association, Western States Affilliate (to B. D.).

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  1. Departments of Immunology and Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, CA, USA

    Becky A. Diebold & Gary M. Bokoch

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Correspondence to Gary M. Bokoch.

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Diebold, B., Bokoch, G. Molecular basis for Rac2 regulation of phagocyte NADPH oxidase. Nat Immunol 2, 211–215 (2001). https://doi.org/10.1038/85259

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