Fig. 1

UV–vis spectra a Q-band region of RoxA as isolated (black), dithionite reduced (2 mM, red), dithionite reduced and subsequently ferricyanide-oxidised (10 mM, green) and a difference spectrum (as isolated minus ferricyanide-oxidised: blue). The difference spectrum shows features at 540 nm and 573 nm after ferricyanide reoxidation. This is characteristic for the loss of haem-bound dioxygen. The minor band at 549 nm in RoxA as isolated represents a small fraction of RoxA molecules with a (deoxygenated) reduced N-terminal haem centre. b Reoxidation of deoxygenated RoxA. UV–vis spectra (Q-band region) of RoxA after reduction with dithionite (twofold molar excess to haem, N₂ atmosphere, red graph) and subsequent reoxidation under a pure O₂-atmosphere. Arrows mark the direction of Q-band changes characteristic for the formation of oxygenated haem (red arrows) by concomitant decrease of reduced haem (black arrow). The green, dark blue, light blue and black lines indicate dithionite-reduced RoxA preparations that were exposed to dioxygen for 5 min, 1 h, 2 h or 4 h, respectively. The pink line shows the difference spectrum of the reaction (black minus red). The black graph after 4 h exposure to oxygen atmosphere basically represents the state as isolated which is oxygenated. For better visualization the spectrum of RoxA as isolated had been, however, omitted. Please note, that this experiment starts with the N-terminal haem in the reduced state and does not require a fully reduced enzyme as it is shown representatively in a (red). c Reaction of RoxA with pyridine. (left scheme): the haem-bound dioxygen molecule in the as isolated state of RoxA (Fe²⁺–O₂) can be substituted by N-heterocyclic compounds, e.g. pyridine (Fe²⁺-Pyr) or imidazole. (right): UV–vis spectra of RoxA as isolated (black), ferricyanide-reoxidised (green), ferricyanide-reoxidised + pyridine-incubated (blue). In contrast to RoxA as isolated, only minor changes in the optical spectrum occur upon pyridine (imidazole) incubation for reoxidised RoxA. This can be explained by binding of pyridine (imidazole) to an oxidised, deoxygenated haem centre. The light blue line shows the difference spectrum of RoxA as isolated minus anaerobically reoxidised RoxA. d RoxA-Wt and RoxA-F317Y after incubation with pyridine. In RoxA-Wt, the “reduction bands” are caused by replacement of dioxygen (Fe²⁺–O₂) with pyridine (Fe²⁺-pyridine) from the N-terminal haem centre. This state resembles the spectrum of reduced RoxA (Fe²⁺). In contrast, RoxA-F317Y as isolated primarily rests in the oxidised state (Fe³⁺). Thus, the binding of pyridine (Fe³⁺-pyridine) does not result in a reduced spectrum. e UV–vis spectroscopic changes of RoxA-Wt and RoxA-F301Y by imidazole or pyridine at room temperature. As isolated (black); incubation with imidazole (red); incubation with pyridine after imidazole incubation (green). In RoxA-Wt, the replacement of dioxygen by imidazole was finished within 100 min at RT, whereas RoxA-F301Y was not affected by imidazole. After addition of 2 mM pyridine, RoxA-F301Y slowly (20 min) converted to the reduced spectrum. For RoxA-Wt almost no additional changes were observed. The total increase of absorption at 549 nm is comparable for both proteins indicating that the portion of proteins with a bound dioxygen molecule is similar for RoxA-Wt and RoxA-F301Y. f Effect of H₂O₂ on the UV–vis spectrum of RoxA. UV–vis spectra of RoxA-Wt (2.5 µM) were recorded before (black line) and every 2 min after the addition of 1 mM H₂O₂. Black arrows indicate the time-dependent changes of haem absorption (every 2 min until endpoint of the haem-destructive effect, indicated by different colours), the red arrow shows the immediate decrease after addition of H₂O₂. Prolonged incubation after the end-point of haem destruction leads to an increase of the 280 nm absorption, but no further decrease of haem absorption indicating an oxidative destruction of the protein backbone (dotted line and arrows). The difference between RoxA-Wt as isolated and after reaction with H₂O₂ at the endpoint of haem-destructive reaction is illustrated on the bottom (brown line), reflecting the spectrum of the formerly oxygenated N-terminal haem centre