Investigating Heme b Biosynthesis from Coproheme

As previously described (Michlits et al., 2020 (link)), H₂O₂ induced conversion of coproheme to heme b was investigated by analyzing the same reaction mix in UV–Vis absorbance and by mass spectrometry. Typically, 1,000 µL enzyme solution in reaction buffer (RB: 50 mM phosphate buffer, pH 7) with around 15 μM apoenzyme and 10 μM coproheme was titrated with subequimolar amounts of hydrogen peroxide (4 mM H₂O₂ stock in RB) in a Cary 60 spectrophotometer (Agilent Technologies). About 10 µl samples from this reaction mix were drawn and directly taken to MS analysis after taking UV–Vis spectra. About 4 µl of the protein solution was directly injected into an LC-ESI-MS system (LC: Agilent 1290 Infinity II UPLC). A gradient from 15 to 80% acetonitrile in 0.1% formic acid (using a Waters BioResolve column (2.1 × 5 mm)) at a flow rate of 400 μl/min was applied (15 min gradient time). Detection was performed with a Q-TOF instrument (Agilent Series 6560 LC-IMS-QTOFMS) equipped with the Jetstream ESI source in positive ion, MS mode (range: 100–3,200 Da). Instrument calibration was performed using the ESI calibration mixture (Agilent Technologies). Data were processed using MassHunter BioConfirm B.08.00 (Agilent Technologies) and the spectrum was deconvoluted by MaxEnt.