The Heme–Lys Cross-Link in Cytochrome P460 Promotes Catalysis by Enforcing Secondary Coordination Sphere Architecture

Abstract

Cytochrome (cyt) P460 is a c-type monoheme enzyme found in ammonia-oxidizing bacteria (AOB) and methanotrophs; additionally, genes encoding it have been found in some pathogenic bacteria. Cyt P460 is defined by a unique post-translational modification to the heme macrocycle, where a lysine (Lys) residue covalently attaches to the 13′ meso carbon of the porphyrin, modifying this heme macrocycle into the enzyme’s eponymous P460 cofactor, similar to the cofactor found in the enzyme hydroxylamine oxidoreductase. This cross-link imbues the protein with unique spectroscopic properties, the most obvious of which is the enzyme’s green color in solution. Cyt P460 from the AOB Nitrosomonas europaea is a homodimeric redox enzyme that produces nitrous oxide (N2O) from 2 equiv of hydroxylamine. Mutation of the Lys cross-link results in spectroscopic features that are more similar to those of standard cyt c′ proteins and renders the enzyme catalytically incompetent for NH2OH oxidation. Recently, the necessity of a second-sphere glutamate (Glu) residue for redox catalysis was established; it plausibly serves as proton relay during the first oxidative half of the catalytic cycle. Herein, we report the first crystal structure of a cross-link deficient cyt P460. This structure shows that the positioning of the catalytically essential Glu changes by approximately 0.8 Å when compared to a cross-linked, catalytically competent cyt P460. It appears that the heme–Lys cross-link affects the relative position of the P460 cofactor with respect to the second-sphere Glu residue, therefore dictating the catalytic competency of the enzyme.

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…The NPs are active for NO binding in the ferric oxidation state (in contrast to H-NOX type NO sensors, which are active in the ferrous state), and use the corresponding ls-{FeNO} 6 adduct for NO transport and delivery. The highly ruffled hemes of rNPs are thought to prevent autoreduction of the ls-{FeNO} 6 complex. − The autoreduction reaction of ferric heme-nitrosyls is observed for globins, for example, and small-molecule model systems, and proceeds by addition of base to the electrophilic NO + ligand in ls-{FeNO} 6 adducts (due to their Fe­(II)–NO + electronic structures; see Section ): where eq is the inverse of the nitrite reduction reaction in eq .…”

Cytochrome P460 Cofactor Maturation Involves a Peroxide- Dependent Post-Translational Modification

“…These similar frequencies suggest that the CLD pro-enzyme remains ruffled, in accord with the structural data obtained for CLD Lys106Leu/Ala131Glu N. sp AL212 cyt P460. (10) Characterization by X-band EPR at 12 K showed that protein is a mixture of S = 5 /2 signals that were consistent with a cross-link containing and a CLD cyt P460 (Fig. 2b).…”

“…Complementary investigations of cyt P460 variants with substituted residues in their distal pockets, combined with a 2.25 Å structure of a cross-link deficient (CLD) cyt P460 variant strongly suggest that the role of the heme-Lys cross-link is to position the cofactor at a sufficiently close distance to a carboxylate group to enable proton transfer during the initial step(s) of NH2OH oxidation. (10,11) While a clear picture is emerging of the functional role of the heme-Lys cross-link, its origins have remained mysterious. The apparent non-necessity of molecular chaperones in recombinant expression of functional, cross-linked cyt P460 has led to speculation that the heme-Lys PTM forms via an autocatalytic process.…”

“…(14,15) Importantly, crystallography shows that ruffling in cyt P460 is not a consequence of the cross-link, but persists even in CLD variants. (10) Given that cyt P460s are produced by obligate aerobic organisms, we hypothesized that the heme-Lys cross-link forms in an oxygen-dependent process-similar to other non-canonical cross-links. ( 16) Further, we have speculated that the structural and electronic requirements for cross-link formation are satisfied by the protein fold and its influence over the bound c-heme cofactor, obviating the need for exogenous maturation factors.…”

A novel catalytic heme cofactor in SfmD with a single thioether bond and a bis-His ligand set revealed by a de novo crystal structural and spectroscopic study

“…Hydroxylamine oxidizing cytochrome P460 has a unique lysine crosslinked c -type monoheme. 68 This heme–Lys crosslink plays a role as a mechanical restraint to bring the secondary sphere glutamate residue close enough to promote catalysis. A hemoglobin, GlbN-A from cyanobacterium Synechococcus , is capable of showing ˙NO dioxygenase activity and has an unusual heme–His crosslink installed by protein posttranslational modification.…”