Spectroscopy of NOX Protein Family Members
All members of the NOX protein family contain a unique b-type cytochrome that mediates the electron transport that characterizes the activity of the multicomponent oxidase complexes. Referred to as cytochrome b558, because of its signature spectral absorbance at 558 nm in reduced-minus-oxidized difference spectroscopy, or cytochrome b(-245), because of its very low midpoint potential of −245 mV at pH 7.0, the protein possesses two stacked inequivalent hemes ligated by pairs of histidine residues in membrane helices h3 and h5. In a flavin-dependent fashion, cytochrome b558 shuttles electrons from cytoplasmic NADPH across membranes to molecular oxygen and thereby generates superoxide anion. By performing reduced-minus-oxidized difference spectroscopy and using the millimolar extinction coefficient, E559–540 nm = 21.6 cm−1 mM−1, one can calculate the amount of cytochrome b558 in intact cells or partially purified membrane preparations. Measurements in samples where cytochrome b558 is relatively high and the presence of unrelated heme-containing proteins low, as in neutrophils, are straightforward. However, low levels of cytochrome b558 expression combined with an abundance of mitochondria and other sources of heme proteins make spectral detection of cytochrome b558 in non-phagocytic cells extremely challenging.
Key wordsCytochrome b558 Cytochrome b(-245) gp91phox NOX proteins Dithionite-reduced-minus-oxidized spectroscopy
The Nauseef lab is supported by National Institute of Health grants AI116546 and AI132335, a Merit Review award from the Veterans Affairs, and use of facilities at the Iowa City Department of Veterans Affairs Medical Center, Iowa City, IA.
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