Double crossed? Structural and computational studies of an unusually crosslinked haem in cytochrome P460.

Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic -type haem where the porphyrin is crosslinked to the protein a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 haem oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460-containing hydroxylamine oxidoreductase utilises a tyrosine crosslink to haem and performs similar chemistry.

Cytochromes P460 and c'-β: exploiting a novel fold for multiple functions.

Two related classes of ligand-binding heme c-containing proteins with a high degree of structural homology have been identified and characterized over recent decades: cytochromes P460 (cyts P460), defined by an unusual heme-lysine cross-link, and cytochromes c'-β (cyts c'-β), containing a canonical c-heme without the lysine cross-link. The shared protein fold of the cyt P460-cyt c'-β superfamily can accommodate a variety of heme environments with entirely different reactivities. On the one hand, cyts P460 with polar distal pockets have been shown to oxidize NHOH to NO and/or NO via proton-coupled electron transfer.

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.

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

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O 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'-β) resembling that of cytochromes P460. A recent structure of cyt c'-β from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site.

Hydroxylamine Complexes of Cytochrome : Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties.

Hydroxylamine (NHOH or HA) is a redox-active nitrogen oxide that occurs as a toxic intermediate in the oxidation of ammonium by nitrifying and methanotrophic bacteria. Within ammonium containing environments, HA is generated by ammonia monooxygenase (nitrifiers) or methane monooxygenase (methanotrophs). Subsequent oxidation of HA is catalyzed by heme proteins, including cytochromes P460 and multiheme hydroxylamine oxidoreductases, the former contributing to emissions of NO, an ozone-depleting greenhouse gas.

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

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

One fold, two functions: cytochrome P460 and cytochrome '-β from the methanotroph (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 '-β (cyt cp-β). Cytochromes P460 are able to convert hydroxylamine to nitrous oxide (NO), a potent greenhouse gas.

Distinguishing Nitro vs Nitrito Coordination in Cytochrome c' Using Vibrational Spectroscopy and Density Functional Theory.

Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest.

Engineering proximal distal heme-NO coordination dinitrosyl dynamics: implications for NO sensor design.

Proximal distal heme-NO coordination is a novel strategy for selective gas response in heme-based NO-sensors. In the case of cytochrome c' (AXCP), formation of a transient distal 6cNO complex is followed by scission of the Fe-His bond and conversion to a proximal 5cNO product a putative dinitrosyl species. Here we show that replacement of the AXCP distal Leu16 residue with smaller or similar sized residues (Ala, Val or Ile) traps the distal 6cNO complex, whereas Leu or Phe residues lead to a proximal 5cNO product with a transient or non-detectable distal 6cNO precursor.

The Dynamics Behind the Affinity: Controlling Heme-Gas Affinity via Geminate Recombination and Heme Propionate Conformation in the NO Carrier Cytochrome c'.

Nitric oxide (NO) sensors are heme proteins which may also bind CO and O. Control of heme-gas affinity and their discrimination are achieved by the structural properties and reactivity of the heme and its distal and proximal environments, leading to several energy barriers. In the bacterial NO sensor cytochrome c' from Alcaligenes xylosoxidans (AXCP), the single Leu16Ala distal mutation boosts the affinity for gas ligands by a remarkable 10-10-fold, transforming AXCP from one of the lowest affinity gas binding proteins to one of the highest.

Cytochromes c': Structure, Reactivity and Relevance to Haem-Based Gas Sensing.

Cytochromes c' are a group of class IIa cytochromes with pentacoordinate haem centres and are found in photosynthetic, denitrifying and methanotrophic bacteria. Their function remains unclear, although roles in nitric oxide (NO) trafficking during denitrification or in cellular defence against nitrosoative stress have been proposed. Cytochromes c' are typically dimeric with each c-type haem-containing monomer folding as a four-α-helix bundle.

Resonance Raman Spectra of Five-Coordinate Heme-Nitrosyl Cytochromes c': Effect of the Proximal Heme-NO Environment.

Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process.

Fingerprinting redox and ligand states in haemprotein crystal structures using resonance Raman spectroscopy.

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochrome c' from Alcaligenes xylosoxidans.

Picosecond binding of the His ligand to four-coordinate heme in cytochrome c': a one-way gate for releasing proximal NO.

We provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome c' from Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant τ(NO) = 7 ps (k(NO) = 1.

A distal pocket Leu residue inhibits the binding of O2 and NO at the distal heme site of cytochrome c'.

Cytochromes c' are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O(2). Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O(2) binding (K(d) ~49 nM) without apparent H-bond stabilization of the Fe-O(2) moiety, as well as an extremely high distal heme-NO affinity (K(d) ~70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing k(on) as well as increasing k(off).

Carbon monoxide poisoning is prevented by the energy costs of conformational changes in gas-binding haemproteins.

Carbon monoxide (CO) is a product of haem metabolism and organisms must evolve strategies to prevent endogenous CO poisoning of haemoproteins. We show that energy costs associated with conformational changes play a key role in preventing irreversible CO binding. AxCYTcp is a member of a family of haem proteins that form stable 5c-NO and 6c-CO complexes but do not form O(2) complexes.

Distal-to-proximal NO conversion in hemoproteins: the role of the proximal pocket.

Hemoproteins play central roles in the formation and utilization of nitric oxide (NO) in cellular signaling, as well as in protection against nitrosative stress. Key to heme-nitrosyl function and reactivity is the Fe coordination number (5 or 6). For (five-coordinate) 5c-NO complexes, the potential for NO to bind on either heme face exists, as in the microbial cytochrome c' from Alcaligenes xylosoxidans (AxCYTcp), which forms a stable proximal 5c-NO complex via a distal six-coordinate NO intermediate and a putative dinitrosyl species.

Activation parameters for heme-NO binding in alcaligenes xylosoxidans cytochrome c': the putative dinitrosyl intermediate forms via a dissociative mechanism.

The bacterial heme protein Alcaligenes xylosoxidans cytochrome c' (AXCP) forms a novel five-coordinate heme-nitrosyl (5c-NO) complex in which NO resides at the proximal heme face in place of the endogenous protein ligand. Intriguingly, AXCP shares NO-binding properties with the eukaryotic NO-sensor, soluble guanylate cyclase (sGC), including 5c-NO formation via two NO-dependent reactions. For both proteins, a model has been proposed in which NO binds to the vacant distal face to form a transient six-coordinate heme-nitrosyl (6c-NO) species, which then converts to a proximal 5c-NO complex via a putative dinitrosyl intermediate.

Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c.

The bacterial heme protein cytochrome ć from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level.

Heterologous overexpression and purification of cytochrome c' from Rhodobacter capsulatus and a mutant (K42E) in the dimerization region. Mutation does not alter oligomerization but impacts the heme iron spin state and nitric oxide binding properties.

Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface.

Accessibility of the distal heme face, rather than Fe-His bond strength, determines the heme-nitrosyl coordination number of cytochromes c': evidence from spectroscopic studies.

The heme coordination chemistry and spectroscopic properties of Rhodobacter capsulatus cytochrome c' (RCCP) have been compared to data from Alcaligenes xylosoxidans (AXCP), with the aim of understanding the basis for their different reactivities with nitric oxide (NO). Whereas ferrous AXCP reacts with NO to form a predominantly five-coordinate heme-nitrosyl complex via a six-coordinate intermediate, RCCP forms an equilibrium mixture of six-coordinate and five-coordinate heme-nitrosyl species in approximately equal proportions. Ferrous RCCP and AXCP both exhibit high Fe-His stretching frequencies (227 and 231 cm(-)(1), respectively), suggesting that factors other than the Fe-His bond strength account for their differences in heme-nitrosyl coordination number.

A novel kinetic trap for NO release from cytochrome c': a possible mechanism for NO release from activated soluble guanylate cyclase.

Flash photolysis studies on the five-coordinate heme nitrosyl of Alcaligenes xylosoxidans cytochrome c' were carried out to investigate the ramifications of its proximal nitrosyl ligand on NO release. Delta absorbance spectra recorded 5 ms after photolysis indicate that approximately 5% of the photolyzed hemes are converted to a five-coordinate high spin ferrous state, revealing that reattachment of the endogenous His ligand is fast enough to trap some of the photolyzed heme. Analysis of NO rebinding suggests that the photolyzed ferrous protein is initially in a strained conformation, which relaxes on a millisecond time scale.

Six- to five-coordinate heme-nitrosyl conversion in cytochrome c' and its relevance to guanylate cyclase.

The 5-coordinate ferrous heme of Alcaligenes xylosoxidans cytochrome c' reacts with NO to form a 6-coordinate nitrosyl intermediate (lambdaSoret at 415 nm) which subsequently converts to a 5-coordinate nitrosyl end product (lambdaSoret at 395 nm) in a rate-determining step. Stopped-flow measurements at pH 8.9, 25 degrees C, yield a rate constant for the formation of the 6-coordinate nitrosyl adduct, k(on) = (4.