Fig. 4

EPR spectra of RoxA. a Effect of hydrogen peroxide on the EPR spectrum of RoxA. EPR spectra were recorded for RoxA as isolated (black) and in the presence of hydrogen peroxide (red). The addition of 10 mM H₂O₂ leads to a rapid increase of a signal at g = 4.28 that indicates unbound Fe³⁺. Since the overall intensity of the EPR signal is not diminished, the unbound Fe³⁺ must have derived from a formerly EPR silent Fe²⁺ species. This illustrates that H₂O₂ reacts exclusively with the N-terminal Fe²⁺ haem, thereby destroying this haem centre. A minor rhombic haem species at g = 2.62, 2.18/2.16 and 1.84 is characteristic for a Fe³⁺–OH ligation (see below, chapter “RoxA retains its activity in the fully oxidised low-spin state”). b EPR spectra of RoxA as isolated (black), reoxidised RoxA (red, dithionite-reduced, then reoxygenated under air) and reoxidised RoxA after incubation with 5 mM pyridine (green). Reoxidised RoxA shows additional intense low- and high-spin ferric EPR-signals from the N-terminal haem group. The high-spin signal originates from an oxidised, fivefold coordinated haem iron (g = 5.97). The new low-spin signal (g = 3.56) is most likely caused by a nearby amino acid residue that takes the position of the previously bound oxygen molecule, establishing a sixfold coordinated haem iron. The addition of pyridine changes the low-spin signals that originate from the amino acid-ligated N-terminal haem-iron, resulting in a new ferric species with a g_z-value of 3.3. c EPR spectra of RoxA in the absence and presence of polyisoprene. RoxA as isolated was incubated for 2 days in buffer (black) or in buffer supplemented with polyisoprene latex for 2 days (red) under anaerobic conditions (N₂). The experiments indicate that a high-spin signal for ferric iron emerges from the EPR-silent ferrous-oxy state in presence of rubber latex under exclusion of dioxygen (red arrow)