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.

Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460

“…We previously showed that cross-link formation in either the WT proenzyme or an Arg44Ala mutant is driven by peroxide, which motivated us to determine whether the Phe41Ala mutant could undergo the same maturation process. 20 Indeed, reaction of 10 μM Phe41Ala cyt P460 with 3 equivalents of Li 2 O 2 (30 μM) resulted in the immediate decay of the 403 nm Soret and isosbestic conversion to a broad Soret centered at 436 nm consistent with a cross-linked ferryl-type compound II product observed previously for WT cyt P460 ( Fig. 4 ).…”

“…S2 † ), which had g -values of 6.02, 5.54, and 1.99 (Table S2 † ). 20 The second component was a more rhombic signal with an E / D = 0.03 and g -values of 6.54, 5.05, and 1.97, consistent with those of CL WT cyt P460. 9 The resonance Raman spectrum obtained with laser excitation at 405 nm was also consistent with previously characterized CLD mutants with an oxidation state marker band ( ν 4 ) at 1367 cm −1 and spin state marker band ( ν 3 ) at 1485 cm −1 (Table S3 † ).…”

“…Comparing this rate to the rate of WT proenzyme did not show any significant changes, with the rate for Phe41Ala cyt P460 being 0.16 ± 0.02 s −1 (μM Li 2 O 2 ) −1 and that of WT proenzyme being 0.13 ± 0.02 s −1 (μM Li 2 O 2 ) −1 . 20 Plotting k obs2 versus Li 2 O 2 concentration also showed a linear dependence with peroxide, albeit a small dependence at only 0.17 min −1 (μM Li 2 O 2 ) −1 . At 40 μM Li 2 O 2 , the observed rate of consumption of the 413 nm intermediate was 2.8 min −1 (μM p460) −1 , whereas the WT proenzyme had a constant rate of 0.42 min −1 (μM p460) −1 .…”

“… 22 We previously used this assay to show that cross-link formation of the WT proenzyme proceeds through a ferryl intermediate. 20 When guaiacol was present during the cross-link reaction of Phe41Ala cyt P460, there was rapid formation of a 470 nm absorbance consistent with guaiacol oxidation (Fig. S4 † ).…”

“…It is instead similar to the recently reported CLD mutant Arg44Ala whose crystal structure showed the uncross-linked Lys forming a salt-bridge with Glu97. 20 While the Arg44Ala mutant did not contain any CL component the Phe41Ala mutant was purified as a mixture, suggesting that cross-link formation may be more facile in the Phe41Ala mutant compared to the Arg44Ala mutant.…”