Cytochrome P460 Cofactor Maturation Proceeds via Peroxide-Dependent Post-translational Modification

Abstract

Cytochrome P460s are heme enzymes that oxidize hydroxylamine to nitrous oxide. They bear specialized "heme P460" cofactors that are cross-linked to their host polypeptides by a post-translationally modified lysine residue. Wild-type N. europaea cytochrome P460 may be isolated as a cross-link-deficient proenzyme following anaerobic overexpression in E. coli. When treated with peroxide, this proenzyme undergoes maturation to active enzyme with spectroscopic and catalytic properties that match wild-type cyt P460. This maturation reactivity requires no chaperones─it is intrinsic to the protein. This behavior extends to the broader cytochrome c'_β superfamily. Accumulated data reveal key contributions from the secondary coordination sphere that enable selective, complete maturation. Spectroscopic data support the intermediacy of a ferryl species along the maturation pathway.

Figure 1.

Known heme-protein cross-links. The purple arrows represent ester bonds to the heme methyl groups, the blue arrows represent cross-links to the vinyl group of the heme, the red arrows represent the tyrosine-heme cross-link in HAO, and the green arrow represents the lysine-heme cross-link in cyt P460. Adapted with permission from Reference . Copyright 2015, Elsevier.

Figure 2.

The heme P460 active site in N. europaea HAO (PDB 4N4N) and cyt P460 (PDB 2JE3). Both are c-type hemes that feature additional covalent attachments. In HAO, a tyrosine residue forms cross-links from the phenolate O to the pyrrole α-carbon and from C_ε to the α-meso-carbon. The heme in cyt P460 contains a cross-link from a lysine N to the γ-meso-carbon.

Figure 3.

Hypothetical mechanism of peroxide-dependent heme-Lys cross-link formation in cyt P460. A) Possible routes for hydroxylation of the meso-carbon followed by B) condensation by lysine.

Figure 4.

Characterization of the anaerobically purified proenzyme and matured proenzyme, which was generated by reaction of the proenzyme with 3 equivalents of Li₂O₂ followed by quenching with sodium dithionite, re-oxidization with [Ru(NH₃)₆]Cl₃, and washing. A) UV/vis absorption spectra of the proenzyme (red) and matured enzyme (black). B) CW X-band EPR spectra collected at 12 K of the proenzyme (red) and matured enzyme (black). Grey traces are the corresponding simulations. C) Resonance Raman spectra obtained via excitation at 405 nm for the proenzyme (red), matured (black), and WT cyt P460 (green). D) Specific activities for WT cyt P460, the proenzyme, and matured proenzyme quantified by the 280 nm extinction coefficient (grey) or the 440 nm extinction coefficient (black). Error bars represent standard deviations of three trials.

Figure 5.

Reaction of 7 μM proenzyme (red) with 3 equivalents (21 μM) of Li₂O₂ at 25 °C. Scans were recorded every 0.3 minutes. The scan immediately following Li₂O₂ addition is shown in black. The final scan is shown in blue, after which the reaction was quenched with Na₂S₂O₄. Time courses of the absorbances corresponding to the proenzyme (404 nm) and product (460 nm) are shown in the inset.

Figure 6.

Continuous-wave X-band EPR spectrum collected at 10 K of the rapid freeze-quench reaction of 200 μM proenzyme with 400 μM Li₂O₂ frozen at 1 second (black) with the simulated spectrum (red). The fit gave g-values of 2.08, 2.01, and 1.98. Oxygen background was removed from the raw experimental spectrum and the cavity was subtracted.

Figure 7.

A) UV/vis spectral time-course for the reaction of the proenzyme with 8 equivalents of Li₂O₂ in the presence of excess (2 mM) guaiacol. The red trace is before Li₂O₂ was added, the black trace was immediately after addition, and the blue trace was 30 minutes after addition. Grey traces represent scans every 0.2 minutes. B) UV/vis spectrum from the reaction after addition of Na₂S₂O₂.

Figure 8.

Fe K-edge XAS XANES region for WT cyt P460 (green) and the reaction of ca. 500 μM proenzyme with 1 equivalent of Li₂O₂ frozen at 1 second (black).

Figure 9.

A) Structure of WT N. europaea cyt P460 (PDB 2JE3) B) Structure of the N. europaea cyt P460 Arg44Ala mutant (PDB 8GAR). C) Overlay of the heme groups from the WT (green) and Arg44Ala (grey) cyt P460 structures, highlighting the change in position of the 6-β-pyrrolic propionate and γ-meso-carbon.

Figure 10.

A) UV/vis spectral time-course for the stopped-flow reaction of 10 μM Arg44Ala with 8 equivalents (80 μM) Li₂O₂. The red trace is the first spectrum after mixing and the blue trace is the final spectrum at 30 seconds. The inset shows the absorbance at 403 nm over time fit to a double exponential. B) UV/vis spectrum comparing as-isolated Arg44Ala (red) and the intermediate formed during the stopped-flow reaction (black).

Figure 11.

UV/vis spectral time-course for the reaction of 8 μM Methylococcus capsulatus WT cyt c′_β (A) or F61K cyt c′_β (B) with 8 equivalents (64 μM) Li₂O₂. The red trace is before the addition of Li₂O₂ and the blue trace is the final spectrum after Li₂O₂ additions. Grey traces represent scans every 0.2 minutes.

Figure 12.

Summary of species observed during peroxide-driven cyt P460 maturation.