One fold, two functions: cytochrome P460 and cytochrome c'-β from the methanotroph Methylococcus capsulatus (Bath)

Abstract

Nature is adept at utilising highly similar protein folds to carry out very different functions, yet the mechanisms by which this functional divergence occurs remain poorly characterised. In certain methanotrophic bacteria, two homologous pentacoordinate c-type heme proteins have been identified: a cytochrome P460 (cyt P460) and a cytochrome c'-β (cyt cp-β). Cytochromes P460 are able to convert hydroxylamine to nitrous oxide (N₂O), a potent greenhouse gas. This reactivity is similar to that of hydroxylamine oxidoreductase (HAO), which is a key enzyme in nitrifying and methanotrophic bacteria. Cyt P460 and HAO both have unusual protein-heme cross-links, formed by a Tyr residue in HAO and a Lys in cyt P460. In contrast, cyts cp-β (the only known cytochromes c' with a β-sheet fold) lack this crosslink and appears to be optimized for binding non-polar molecules (including NO and CO) without enzymatic conversion. Our bioinformatics analysis supports the proposal that cyt cp-β may have evolved from cyt P460 via a gene duplication event. Using high-resolution X-ray crystallography, UV-visible absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopy, we have characterized the overall protein folding and active site structures of cyt cp-β and cyt P460 from the obligate methanotroph, Methylococcus capsulatus (Bath). These proteins display a similar β-sheet protein fold, together with a pattern of changes to the heme pocket regions and localised tertiary structure that have converted a hydroxylamine oxidizing enzyme into a gas-binding protein. Structural comparisons provide insights relevant to enzyme redesign for synthetic enzymology and engineering of gas sensor proteins. We also show the widespread occurrence of cyts cp-β and characterise their phylogeny.

Fig. 1. The homodimeric structures of (A) McP460 and (B) McCP-β showing the predominantly β-sheet fold; (C) the heme environment in McP460, note the distal water ligand and hydrophilic, charged pocket with several Arg and Asp residues in a position to interact with bound substrates; (D) the heme environment of McCP-β showing the hydrogen bonding between the proximal His 123 and the backbone carbonyl of Tyr 99. The distal pocket is devoid of water molecules and formed of hydrophobic residues including the ‘Phe Cap’ of Phe 32 and Phe 61; (E) superposition of McCP-β (blue) and McP460 (coral) overall structures; (F) superimposition of the heme environments of McCP-β (blue) and McP460 (coral).

Fig. 2. RR spectra of ferric and ferrous McP460 in the high-frequency (a) and low frequency (b) regions; RR spectra of ferric and ferrous McCP-β in the high-frequency (c) and low-frequency (d) regions. Data obtained at room temperature using either 407 nm or 442 nm excitation as indicated. Solutions contained ∼150 μM protein (in haem) in pH 7.0 buffer (50 mM MOPS).

Fig. 3. CD spectra (a) of McCP-β (dashed line) and McP460 (solid line). (b) Thermal denaturation curves for McCP-β (closed circles) and McP460 (open circles) as measured by CD. Representative normalized raw data plots are shown at the temperature interval of 2.5 °C.

Fig. 4. Maximum likelihood tree of cyt cp-α (blue circles), cyt cp-β (purple circles) and cyt P460 (green triangles) sequences. The cyt cp-α genes are on a separate branch to the cyt cp-β and the cyts P460 genes. The cyt P460 family displays a paraphyletic structure, with a monophyletic cyt cp-β nested within suggesting that the cyt cp-β evolved from the cyt P460. Image was prepared using Mega7.

Fig. 5. Out-of-plane displacements (minimal basis) for the hemes of McCP-βl (6HIH), McP460 (; 6HIU), NsALP460 (; 6AMG), N. europaea P460 (J2E3) and the P460 heme of N. europaea HAO (4FAS). Out of plane distortions are characterized in terms of displacements along the normal coordinates of the D_4h-symmetric porphyrin macrocycle. NsALP460 and NeP460 display much higher overall distortions to both M. capsulatus (Bath) cytochromes. McP460 displays a high amount of ruffling (B1u) which has been proposed to be important in the function of the P460 cytochromes. In comparison to the other two cytochromes P460, McP460 does not have an equal amount of ruffling and saddling, a pattern that is more similar to that of the P460 heme of N. europaea HAO. McCP-β correspondingly does not display a high level of ruffling, although it does demonstrate a large amount of saddling (B2u). See also Table S5.