Conformational rigidity of cytochrome c'-α from a thermophile is associated with slow NO binding.

Cytochromes c'-α are nitric oxide (NO)-binding heme proteins derived from bacteria that can thrive in a wide range of temperature environments. Studies of mesophilic Alcaligenes xylosoxidans cytochrome c'-α (AxCP-α) have revealed an unusual NO-binding mechanism involving both heme faces, in which NO first binds to form a distal hexa-coordinate Fe(II)-NO (6cNO) intermediate and then displaces the proximal His to form a proximal penta-coordinate Fe(II)-NO (5cNO) final product. Here, we characterize a thermally stable cytochrome c'-α from thermophilic Hydrogenophilus thermoluteolus (PhCP-α) to understand how protein thermal stability affects NO binding. Electron paramagnetic and resonance Raman spectroscopies reveal the formation of a PhCP-α 5cNO product, with time-resolved (stopped-flow) UV-vis absorbance indicating the involvement of a 6cNO intermediate. Relative to AxCP-α, the rates of 6cNO and 5cNO formation in PhCP-α are ∼11- and ∼13-fold lower, respectively. Notably, x-ray crystal structures of PhCP-α in the presence and absence of NO suggest that the sluggish formation of the proximal 5cNO product results from conformational rigidity: the Arg-132 residue (adjacent to the proximal His ligand) is held in place by a salt bridge between Arg-75 and Glu-135 (an interaction not present in AxCP-α or a psychrophilic counterpart). Overall, our data provide fresh insights into structural factors controlling NO binding in heme proteins, including 5cNO complexes relevant to eukaryotic NO sensors.

A heme pocket aromatic quadrupole modulates gas binding to cytochrome c'-ß: Implications for NO sensors.

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-ß) resembling that of cytochromes P460. A recent structure of cyt c'-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

Crystal structure of thermally stable homodimeric cytochrome c'-ß from Thermus thermophilus.

Cytochrome c'-ß is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel ß-sheet fold. Here, the crystal structure of cytochrome c'-ß from the thermophilic Thermus thermophilus (TTCP-ß) is reported at 1.74 Šresolution. TTCP-ß has a typical antiparallel ß-sheet fold similar to that of cytochrome c'-ß from the moderately thermophilic Methylococcus capsulatus (MCCP-ß). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-ß and MCCP-ß, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-ß exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-ß. Mutational analysis reveals that the increased homodimeric interface area of TTCP-ß contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-ß loop regions, possibly contribute to the rigid loop structure compared with MCCP-ß, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-ß, including TTCP-ß, are optimized for function under the high-temperature conditions in which the source organisms live.

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

[This corrects the article DOI: 10.1039/C8SC05210G.].

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

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'-ß (cyt cp-ß). 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-ß (the only known cytochromes c' with a ß-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-ß 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-ß and cyt P460 from the obligate methanotroph, Methylococcus capsulatus (Bath). These proteins display a similar ß-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-ß and characterise their phylogeny.

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

[This corrects the article DOI: 10.1039/C8SC05210G.].