Quantifying Heme–Protein Maturation from Ratiometric Fluorescence Lifetime Measurements on the Single Fluorophore in Its GFP Fusion

Protein maturation by heme insertion is a common post-translation modification of key biological importance. Nonetheless, where and when this maturation occurs in eukaryotic cells remain unknown for most heme proteins. Here, we demonstrate for the first time that the maturation of a chromosomally expressed, endogenous heme protein fused to a green fluorescent protein (GFP) can be tracked in live cells. Selecting yeast cytochrome c peroxidase (Ccp1) as our model heme-binding protein, we first characterized the emission in vitro of recombinant Ccp1–GFP with GFP fused C-terminally to Ccp1 by the linker GRRIPGLIN. Time-correlated single-photon counting reveals a single fluorescence lifetime for heme-free apoCcp1–GFP, τ₀ = 2.84 ± 0.01 ns. Heme bound to Ccp1 only partially quenches GFP fluorescence since holoCcp1–GFP exhibits two lifetimes, τ₁ = 0.95 ± 0.02 and τ₂ = 2.46 ± 0.03 ns with fractional amplitudes a₁ = 38 ± 1.5% and a₂ = 62 ± 1.5%. Also, τ and a are independent of Ccp1–GFP concentration and solution pH between 5.5 and 8.0, and a standard plot of a₁ vs % holoCcp1–GFP in mixtures with apoCcp1–GFP is linear, establishing that the fraction of Ccp1–GFP with heme bound can be determined from a₁. Fluorescence lifetime imaging microscopy (FLIM) of live yeast cells chromosomally expressing the same Ccp1–GFP fusion revealed 30% holoCcp1–GFP (i.e., mature Ccp1) and 70% apoCcp1–GFP in agreement with biochemical measurements on cell lysates. Thus, ratiometric fluorescence lifetime measurements offer promise for probing heme–protein maturation in live cells, and we can dispense with the reference fluorophore required for ratiometric intensity-based measurements.