Invariant local conformation in p22phox p.Y72H polymorphisms suggested by mass spectral analysis of crosslinked human neutrophil flavocytochrome b

The NADPH oxidase of phagocytic leukocytes generates superoxide that plays a critical role in innate immunity and inflammatory responses. The integral membrane protein flavocytochrome b (Cyt b, a.k.a. cytochrome b558/559) is the catalytic core of the complex and serves as a prototype for homologs important in regulating signaling networks in a wide variety of animal and plant cells. Our analysis identifies a naturally-occurring Tyr72/His72 polymorphism (p.Y72H) in the p22phox subunit of Cyt b at the protein level that has been recognized at the nucleotide level (c.214T > C, formerly C242T) and implicated in cardiovascular disease. In the present study, Cyt b was isolated from human neutrophils and reacted with chemical crosslinkers for subsequent structure analysis by MALDI mass spectrometry. Following mild chemical modification of Cyt b with two pairs of isotopically-differentiated lysine crosslinkers: BS2G-d0/d4 and BS3-d0/d4, the reaction mixtures were digested with trypsin and purified on C18ZipTips to generate samples for mass analysis. MALDI analysis of tryptic digests from each of the above reactions revealed a series of masses that could be assigned to p22phox residues 68–85, assuming an intra-molecular crosslink between Lys71 and Lys78. In addition to the 30 ppm mass accuracy obtained with internal mass calibration, increased confidence in the assignment of the crosslinks was provided by the presence of the diagnostic mass patterns resulting from the isotopically-differentiated crosslinking reagent pairs and the Tyr72/His72 p22phox polymorphisms in the crosslinked peptides. This work identifies a novel, low-resolution distance constraint in p22phox and suggests that the medically-relevant p.Y72H polymorphism has an invariant structural motif in this region. Because position 72 in p22phox lies outside regions identified as interactive with other oxidase components, the structural invariance also provides additional support for maturational differences as the source of the wide variation in observed reactive oxygen species production by cells expressing p.Y72H.

► Mass Spectrometry/Intra-molecular Cross-linking of Flavocytochrome b p22phox subunit. ► Intra/Intermolecular Flavocytochrome b Crosslinking with BS2G-d0/d4 and BS3-d0/d4. ► Epsilon amino groups of Lys71 and Lys78 of p22phox are approximately 7–12 Å apart. ► Local conformation at polymorphisms p.Y72H of p22phox are invariant. ► Haplogroup ROS functional differences may arise from p22phox differential expression.