The Eponymous Cofactors in Cytochrome P460s from Ammonia-Oxidizing Bacteria Are Iron Porphyrinoids Whose Macrocycles Are Dibasic

Abstract

The enzymes hydroxylamine oxidoreductase and cytochrome (cyt) P460 contain related unconventional "heme P460" cofactors. These cofactors are unusual in their inclusion of nonstandard cross-links between amino acid side chains and the heme macrocycle. Mutagenesis studies performed on the Nitrosomonas europaea cyt P460 that remove its lysine-heme cross-link show that the cross-link is key to defining the spectroscopic properties and kinetic competence of the enzyme. However, exactly how this cross-link confers these features remains unclear. Here we report the 1.45 Å crystal structure of cyt P460 from Nitrosomonas sp. AL212 and conclude that the cross-link does not lead to a change in hybridization of the heme carbon participating in the cross-link but rather enforces structural distortions to the macrocycle away from planarity. Time-dependent density functional theory coupled to experimental structural and spectroscopic analysis suggest that this geometric distortion is sufficient to define the spectroscopic properties of the heme P460 cofactor and provide clues toward establishing a relationship between heme P460 electronic structure and function.

Figure 1.

Heme P460 cofactors from N. europaea (a) hydroxylamine oxidoreductase (PDBID: 4FAS) and (b) cytochrome P460 (PDBID: 2JE3).

Figure 2.

A single sp² meso C in a porphyrin backbone can give rise to either isoporphyrin or phlorin electronic configurations.

Figure 3.

(a) UV-vis absorption spectra of resting Fe^III (black), {FeNO} (red), and {FeNO} (gray) forms of Nitrosomonas sp. AL212 cyt P460. (b) 10 K X-band (9.40 GHz) EPR spectrum of Fe^III Nitrosomonas sp. AL212 cyt P460 (black) recorded at 633 μW microwave power. The corresponding SpinCount simulation is shown in red.

Figure 4.

Crystal structure of Nitrosomonas sp. AL212 Fe^III cyt P460 (PDB ID 6AMG). Subunit A shown in cyan, subunit B shown in purple. The heme P460 cofactors are shown in green.

Figure 5.

Cofactor binding pocket of Nitrosomonas sp. AL212 cyt P460 depicted using (a) sticks or (b) space-filling spheres. The 2F_o-F_c simulated annealing omit map at a level of 2.5 σ is displayed for the loops that cover the distal side of the heme P460 cofactor. Subunit A shown in cyan, subunit B shown in purple. The heme P460 cofactors are shown in green.

Figure 6.

2F_o-F_c simulated annealing omit map shown at 2.5 σ and corresponding stick representation of the heme P460 cofactor of subunit A from Nitrosomonas sp. AL212 Fe^III cyt P460. The inset shows the 13’ meso C participating in the cross-link with the Lys106 sidechain and corresponding metrical parameters about this atom.

Figure 7.

(a) Experimental and (b) TDDFT-calculated (B3LYP/ZORA-def2-TZVP(-f) with CP(PPP) on Fe) UV-vis absorption spectra of horse heart metmyoglobin and Fe^III Nitrosomonas sp. AL212 cyt P460. In (b), spectra are calculated for isoporphyrin, porphyrin, and phlorin macrocycle configurations.

Figure 8.

TDDFT-calculated (B3LYP/ZORA-def2-TZVP(-f) with CP(PPP) on Fe) UV-vis absorption spectra of horse heart metmyoglobin and Fe^III Nitrosomonas sp. AL212 cyt P460 variants where the 13’ meso C is bound to H, C (from RCH2), or N (from RNH).

Figure 9.

(a) Simplified, top down depiction of the frontier molecular orbitals of a porphyrin π-system modified to show the meso-C cross-link and labeled with corresponding D_4h irreducible representations, (b) Quasi-restricted orbital (QROs) plots depicting the porphyrin π-system molecular orbitals of the Fe^III cyt P460 cofactor with Lys N lone-pair admixture. Calculations were carried out using B3LYP/ZORA-def2-TZVP(-f) with CP(PPP) on Fe, and orbitals are plotted at an isovalue of 0.03 au.

Figure 10.

Simplified energy diagram depicting frontier porphyrin π-system molecular orbitals of the Fe^III cyt P460 cofactor as a dibasic porphyrin with Lys cross-link (meso C-N) and dibasic porphyrin variants where the cross-link is modified to a meso C-C linkage or removed entirely (meso C-H). The red lines indicate molecular orbitals whose energies are influenced by orbital admixture from the cross-link.

Scheme 1.

Atom numbering used for the Nitrosomonas sp. AL212 cyt P460 cofactor.