Controlling a burn: outer-sphere gating of hydroxylamine oxidation by a distal base in cytochrome P460.

Ammonia oxidizing bacteria (AOB) use the cytotoxic, energetic molecule hydroxylamine (NH2OH) as a source of reducing equivalents for cellular respiration. Despite disproportionation or violent decomposition being typical outcomes of reactions of NH2OH with iron, AOB and anammox heme P460 proteins including cytochrome (cyt) P460 and hydroxylamine oxidoreductase (HAO) effect controlled, stepwise oxidation of NH2OH to nitric oxide (NO). Curiously, a recently characterized cyt P460 variant from the AOB Nitrosomonas sp. AL212 is able to form all intermediates of cyt P460 catalysis, but is nevertheless incompetent for NH2OH oxidation. We now show via site-directed mutagenesis, activity assays, spectroscopy, and structural biology that this lack of activity is attributable to the absence of a critical basic glutamate residue in the distal pocket above the heme P460 cofactor. This substitution is the only distinguishing characteristic of a protein that is otherwise effectively structurally and spectroscopically identical to an active variant. This highlights and reinforces a fundamental principal of metalloenzymology: metallocofactor inner-sphere geometric and electronic structures are in many cases insufficient for imbuing reactivity; a precisely defined outer coordination sphere contributed by the polypeptide matrix can be the key differentiator between a metalloenzyme and an unreactive metalloprotein.

Dramatic Electronic Perturbations of CuA Centers via Subtle Geometric Changes.

CuA is a binuclear copper site acting as electron entry port in terminal heme-copper oxidases. In the oxidized form, CuA is a mixed valence pair whose electronic structure can be described using a potential energy surface with two minima, σu* and πu, that are variably populated at room temperature. We report that mutations in the first and second coordination spheres of the binuclear metallocofactor can be combined in an additive manner to tune the energy gap and, thus, the relative populations of the two lowest-lying states. A series of designed mutants span σu*/πu energy gaps ranging from 900 to 13 cm-1. The smallest gap corresponds to a variant with an effectively degenerate ground state. All engineered sites preserve the mixed-valence character of this metal center and the electron transfer functionality. An increase of the Cu-Cu distance less than 0.06 Å modifies the σu*/πu energy gap by almost 2 orders of magnitude, with longer distances eliciting a larger population of the πu state. This scenario offers a stark contrast to synthetic systems, as model compounds require a lengthening of 0.5 Å in the Cu-Cu distance to stabilize the πu state. These findings show that the tight control of the protein environment allows drastic perturbations in the electronic structure of CuA sites with minor geometric changes.

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

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.