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.
Highlights
Understanding the means by which enzymes can utilise a common fold and structural motifs to evolve very different functions is essential for synthetic biology and rational enzyme design for biotechnology
In this study we present high resolution crystal structures of McCP-b and McP460 in their as-isolated states, representing the rst crystal structure of any cyt cp-b, and the rst for a methanotrophic cyt P460
The crystal structure of as-isolated McP460 was determined to 1.36 Aresolution (Fig. 1, Table 1) with residues 19 to 161 observed in the electron density maps
Summary
Understanding the means by which enzymes can utilise a common fold and structural motifs to evolve very different functions is essential for synthetic biology and rational enzyme design for biotechnology. Edge Article a result of ammonia monooxygenase or methane monooxygenase activity in ammonia oxidising and methane oxidising bacteria (AOB or MOB) respectively.[8] Hydroxylamine oxidation occurs in the unrelated hydroxylamine oxidoreductases (HAO) which, in addition to containing multiple heme centers, have a different type of P460 porphyrin cross-link involving a conserved Tyr, as well as very different protein folds.[9,10,11] The HAO P460 heme pocket contains Tyr, Asp and His residues implicated in reactivity.[9,10,11] Structures of cyt P460 12 and HAO9,10 from the ammonia oxidising bacterium Nitrosomonas europea, as well as cyt P460 from Nitrosomonas sp. AL212 (NsALP460) have been the focus of several recent spectroscopic studies.[12,13,14,15] A relationship between heme P460 character, the protein crosslink, and distortion of the heme geometry away from planarity has recently been proposed.[13]
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