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Cascade Blue acetyl azide is an amine reactive compound with spectral properties ideally suited for fluorescence resonance energy transfer (FRET) studies in which heme prosthetic groups serve as acceptors. To demonstrate utility of the Cascade Blue-heme spectroscopic ruler, cytochrome c was employed as a test case to calibrate distance measurements obtained from FRET analysis. Following modification, stoichiometrically labeled cytochrome c was digested with trypsin and derivatized fragments were analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry to identify Lys25 as the predominant site of covalent modification. FRET analysis on derivatized protein demonstrated nearly complete quenching of Cascade Blue fluorescence, indicating the labeled lysine residue to reside within 30 A of the heme prosthetic group. Sodium dodecyl sulfate (SDS) denaturation resulted in an approximately 28% recovery of fluorescence, demonstrating the utility of this donor-acceptor pair for evaluating distance changes of 30-90 A. Modeling the Cascade Blue donor molecule onto Lys25 of a cytochrome c NMR structure confirmed a distance of < or =30 A from the heme acceptor, as determined by FRET analysis. Further modeling of the SDS-denatured state as an extended chain suggested a maximum separation distance of 45 A, also consistent with results derived from FRET analysis.