Diversifying the functions of heme proteins with non-porphyrin cofactors.

Heme proteins perform diverse biochemical functions using a single iron porphyrin cofactor. This versatility makes them attractive platforms for the development of new functional proteins. While directed evolution and metal substitution have expanded the properties, reactivity, and applications of heme proteins, the incorporation of porphyrin analogs remains an underexplored approach. This review discusses the replacement of heme with non-porphyrin cofactors, such as porphycene, corrole, tetradehydrocorrin, phthalocyanine, and salophen, and the attendant properties of these conjugates. While structurally similar, each ligand exhibits distinct optical and redox properties, as well as unique chemical reactivity. These hybrids serve as model systems to elucidate the effects of the protein environment on the electronic structure, redox potentials, optical properties, or other features of the porphyrin analog. Protein encapsulation can confer distinct chemical reactivity or selectivity of artificial metalloenzymes that cannot be achieved with the small molecule catalyst alone. Additionally, these conjugates can interfere with heme acquisition and uptake in pathogenic bacteria, providing an inroad to innovative antibiotic strategies. Together, these examples illustrate the diverse functionality that can be achieved by cofactor substitution. The further expansion of this approach will access unexplored chemical space, enabling the development of superior catalysts and the creation of heme proteins with emergent properties.

Ag(III)···Ag(III) Argentophilic Interaction in a Cofacial Corrole Dyad.

Metallophilic interactions between closed-shell metal centers are exemplified by d10 ions, with Au(I) aurophilic interactions as the archetype. Such an interaction extends to d8 species, and examples involving Au(III) are prevalent. Conversely, Ag(III) argentophilic interactions are uncommon. Here, we identify argentophilic interactions in silver corroles, which are authentic Ag(III) species. The crystal structure of a monomeric silver corrole is a dimer in the solid state, and the macrocycle exhibits an atypical domed conformation. In order to evaluate whether this represents an authentic metallophilic interaction or a crystal-packing artifact, the analogous cofacial or "pacman" corrole was prepared. The conformation of the monomer was recapitulated in the silver pacman corrole, exhibiting a short 3.67 Å distance between metal centers and a significant compression of the xanthene backbone. Theoretical calculations support the presence of a rare Ag(III)···Ag(III) argentophilic interaction in the pacman complex.

Solvent-Induced Spin-State Change in Copper Corroles.

The electronic structure of copper corroles has been a topic of debate and revision since the advent of corrole chemistry. The ground state of these compounds is best described as an antiferromagnetically coupled Cu(II) corrole radical cation. In coordinating solvents, these molecules become paramagnetic, and this is often accompanied by a color change. The underlying chemistry of these solvent-induced properties is currently unknown. Here, we show that a coordinating solvent, such as pyridine, induces a change in the ground spin state from an antiferromagnetically coupled Cu(II) corrole radical cation to a ferromagnetically coupled triplet. Over time, the triplet reacts to produce a species with spectral signatures that are characteristic of the one-electron-reduced Cu(II) corrole. These observations account for the solvent-induced paramagnetism and the associated color changes that have been observed for copper corroles in coordinating solvents.

Corrole-protein interactions in H-NOX and HasA.

Replacing the native porphyrin cofactor in haem proteins has led to the development of novel designer proteins for a variety of applications. In most cases, haem analogues bind in a way that is comparable to the iron porphyrin, but this is not necessarily the case for complexes bearing non-exchangeable ligands. This study probes how a P[double bond, length as m-dash]O corrole binds to functionally disparate hemoproteins: a haem-dependent oxygen sensor (H-NOX) and a haem-scavenging protein (HasA). The results demonstrate that the protein-cofactor interactions are distinct from the native, haem-bound holoprotein. In H-NOX, the P[double bond, length as m-dash]O unit primarily hydrogen bonds with the haem-ligating histidine (H102), rather than the hydrogen-bonding network that stabilises the Fe(ii)-O2 complex in the native protein. In the absence of H102, the protein is still able to bind the corrole, albeit at reduced levels. Molecular dynamics simulations were utilised to determine the flexibility of apo H-NOX and revealed the coupled motion of key residues necessary for corrole binding. In the case of HasA, the P[double bond, length as m-dash]O unit does not primarily interact with either the haem-ligating histidine (H32) or tyrosine (Y75). Instead, histidine 83, the hydrogen-bonding partner for Y75, is critical for P[double bond, length as m-dash]O corrole binding. The conformation of HasA is interrogated by site-specifically labelling the protein and exploiting Förster resonance energy transfer (FRET) to determine the dye-cofactor distance. HasA reconstituted with the P[double bond, length as m-dash]O corrole exhibits an extended, apo-like conformation. Together, these results demonstrate that non-natural cofactors can bind to proteins in unexpected ways and highlight the need to uncover these interactions for the further development of designer haem proteins.

Ratiometric Oxygen Sensing with H-NOX Protein Conjugates.

Ratiometric sensors are self-referencing constructs that are functional in cells and tissues, and the read-out is independent of sensor concentration. One strategy for ratiometric sensing is to utilize two-color emission, where one component possesses analyte-dependent emission and the other is independent of analyte concentration, serving as an internal standard. In this way, the intensity ratio of the two components is a quantitative measure of the analyte. In this study, protein-based ratiometric oxygen sensors are prepared using the heme nitric oxide/oxygen-binding protein (H-NOX) from the thermophilic bacterium Caldanaerobacter subterraneus. The native heme cofactor is replaced with a Pd(II) or Pt(II) porphyrin as the oxygen-responsive phosphor. Mutagenesis is performed to incorporate a cysteine residue on the protein surface for thiol/maleimide coupling of the oxygen-insensitive dye, which serves as a Förster resonance energy transfer (FRET) donor for the porphyrin. While both Pd(II)- and Pt(II)-based sensors are responsive over biologically relevant ranges, the Pd sensor exhibits greater sensitivity at lower oxygen concentrations. Together, these sensors represent a new class of protein-based ratiometric oxygen sensors, and the modular platform allows the oxygen sensitivity to be tailored for a specific application. This proof-of-principle study has identified the key considerations and optimal methodologies to develop and subsequently refine protein-based ratiometric oxygen sensors.

Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues.

Heme proteins have proven to be a convenient platform for the development of designer proteins with novel functionalities. This is achieved by substituting the native iron porphyrin cofactor with a heme analogue that possesses the desired properties. Replacing the iron center of the porphyrin with another metal provides one inroad to novel protein function. A less explored approach is substitution of the porphyrin cofactor with an alternative tetrapyrrole macrocycle or a related ligand. In general, these ligands exhibit chemical properties and reactivity that are distinct from those of porphyrins. While these techniques have most prominently been utilized to develop artificial metalloenzymes, there are many other applications of this methodology to problems in biochemistry, health, and medicine. Incorporation of synthetic cofactors into protein environments represents a facile way to impart water solubility and biocompatibility. It circumvents the laborious synthesis of water-soluble cofactors, which often introduces substantial charge that leads to undesired bioaccumulation. To this end, the incorporation of unnatural cofactors in heme proteins has enabled the development of designer proteins as optical oxygen sensors, MRI contrast agents, spectroscopic probes, tools to interrogate protein function, antibiotics, and fluorescent proteins.Incorporation of an artificial cofactor is frequently accomplished by denaturing the holoprotein with removal of the heme; the refolded apoprotein is then reconstituted with the artificial cofactor. This process often results in substantial protein loss and does not necessarily guarantee that the refolded protein adopts the native structure. To circumvent these issues, our laboratory has pioneered the use of the RP523 strain of E. coli to incorporate artificial cofactors into heme proteins using expression-based methods. This strain lacks the ability to biosynthesize heme, and the bacterial cell wall is permeable to heme and related molecules. In this way, heme analogues supplemented in the growth medium are incorporated into heme proteins. This approach can also be leveraged for the direct expression of the apoprotein for subsequent reconstitution.These methodologies have been exploited to incorporate non-native cofactors into heme proteins that are resistant to harsh environmental conditions: the heme nitric oxide/oxygen binding protein (H-NOX) from Caldanaerobacter subterraneus (Cs) and the heme acquisition system protein A (HasA) from Pseudomonas aeruginosa (Pa). The exceptional stability of these proteins makes them ideal scaffolds for biomedical applications. Optical oxygen sensing has been accomplished using a phosphorescent ruthenium porphyrin as the artificial heme cofactor. Paramagnetic manganese and gadolinium porphyrins yield high-relaxivity, protein-based MRI contrast agents. A fluorescent phosphorus corrole serves as a heme analogue to produce fluorescent proteins. Iron complexes of nonporphyrin cofactors bound to HasA inhibit the growth of pathogenic bacteria. Moreover, HasA can deliver a gallium phthalocyanine into the bacterial cytosol to serve as a sensitizer for photochemical sterilization. Together, these examples illustrate the potential for designer heme proteins to address burgeoning problems in the areas of health and medicine. The concepts and methodologies presented in this Account can be extended to the development of next-generation biomedical sensing and imaging agents to identify and quantify clinically relevant metabolites and other key disease biomarkers.

Corrole-Substituted Fluorescent Heme Proteins.

Although fluorescent proteins have been utilized for a variety of biological applications, they have several optical limitations, namely weak red and near-infrared emission and exceptionally broad (>200 nm) emission profiles. The photophysical properties of fluorescent proteins can be enhanced through the incorporation of novel cofactors with the desired properties into a stable protein scaffold. To this end, a fluorescent phosphorus corrole that is structurally similar to the native heme cofactor is incorporated into two exceptionally stable heme proteins: H-NOX from Caldanaerobacter subterraneus and heme acquisition system protein A (HasA) from Pseudomonas aeruginosa. These yellow-orange emitting protein conjugates are examined by steady-state and time-resolved optical spectroscopy. The HasA conjugate exhibits enhanced fluorescence, whereas emission from the H-NOX conjugate is quenched relative to the free corrole. Despite the low fluorescence quantum yields, these corrole-substituted proteins exhibit more intense fluorescence in a narrower spectral profile than traditional fluorescent proteins that emit in the same spectral window. This study demonstrates that fluorescent corrole complexes are readily incorporated into heme proteins and provides an inroad for the development of novel fluorescent proteins.

Multielectron C-H photoactivation with an Sb(v) oxo corrole.

Pnictogen complexes are ideal for mediating multi-electron chemical reactions in two-electron steps. We report an Sb(v) bis-µ-oxo corrole that photochemically oxidises the C-H bonds of organic substrates. In the case of toluene, the substrate is oxidised to benzaldehyde, a rare example of a four-electron photoreaction.

Halogen Photoelimination from SbV Dihalide Corroles.

Main-group p-block metals are ideally suited for mediating two-electron reactions because they cycle between M n and M n+2 redox states, as the one-electron state is thermodynamically unstable. Here, we report the synthesis and structure of an SbIII corrole and its SbVX2 (X = Cl, Br) congeners. SbIII sits above the corrole ring, whereas SbV resides in the corrole centroid. Electrochemistry suggests interconversion between the SbIII and SbVX2 species. TD-DFT calculations indicate a HOMO → LUMO+2 parentage for excited states in the Soret spectral region that have significant antibonding character with respect to the Sb-X fragment. The photochemistry of 2 and 3 in THF is consistent with the computational results, as steady-state photolysis at wavelengths coincident with the Soret absorption of SbVX2 corrole lead to its clean conversion to the SbIII corrole. This ability to photoactivate the Sb-X bond reflects the proclivity of the pnictogens to rely on the PnIII/V couple to drive the two-electron photochemistry of M-X bond activation, an essential transformation needed to develop HX-splitting cycles.

Gold Corroles as Near-IR Phosphors for Oxygen Sensing.

The triplet state of gold(III) corroles is exploited for optical oxygen sensing. We report intense phosphorescence for gold(III) corroles in the near-IR, an optical window that is ideal for tissue transparency. Moreover, the triplet excited-state emission exhibits significant changes in intensity and lifetime over the 0-160 Torr O2 pressure range. This renders these compounds sensitive at biologically relevant pressures and overcomes the spectral limitations of palladium and platinum porphyrins for oxygen sensing in biology.

Electronic Structure of Copper Corroles.

The ground state electronic structure of copper corroles has been a topic of debate and revision since the advent of corrole chemistry. Computational studies formulate neutral Cu corroles with an antiferromagnetically coupled Cu(II) corrole radical cation ground state. X-ray photoelectron spectroscopy, EPR, and magnetometry support this assignment. For comparison, Cu(II) isocorrole and [TBA][Cu(CF3)4] were studied as authentic Cu(II) and Cu(III) samples, respectively. In addition, the one-electron reduction and one-electron oxidation processes are both ligand-based, demonstrating that the Cu(II) centre is retained in these derivatives. These observations underscore ligand non-innocence in copper corrole complexes.

Comparison of self-assembled and micelle encapsulated QD chemosensor constructs for biological sensing.

Whereas a variety of covalent conjugation strategies have been utilized to prepare quantum dot (QD)-based nanosensors, supramolecular approaches of self-assembly have been underexplored. A major advantage of self-assembly is the ability to circumvent laborious synthetic efforts attendant to covalent conjugation of a chemosensor to functionalized QDs. Here, we combine a CdSe/ZnS core-shell QD with gold(III) corroles using both self-assembly and micelle encapsulation to form QD nanosensors. Appreciable spectral overlap between QD emission and corrole absorption results in efficient Förster resonance energy transfer (FRET), which may be initiated by one- or two-photon excitation. The triplet state of the gold(III) corroles is quenched by molecular oxygen, enabling these constructs to function as optical O2 sensors, which is useful for the metabolic profiling of tumours. The photophysical properties, including QD and corrole lifetimes, FRET efficiency, and O2 sensitivity, have been determined for each construct. The relative merits of each conjugation strategy are assessed with regard to their implementation as sensors.

Micelle-Encapsulated Quantum Dot-Porphyrin Assemblies as in Vivo Two-Photon Oxygen Sensors.

Micelles have been employed to encapsulate the supramolecular assembly of quantum dots with palladium(II) porphyrins for the quantification of O2 levels in aqueous media and in vivo. Förster resonance energy transfer from the quantum dot (QD) to the palladium porphyrin provides a means for signal transduction under both one- and two-photon excitation. The palladium porphyrins are sensitive to O2 concentrations in the range of 0-160 Torr. The micelle-encapsulated QD-porphyrin assemblies have been employed for in vivo multiphoton imaging and lifetime-based oxygen measurements in mice with chronic dorsal skinfold chambers or cranial windows. Our results establish the utility of the QD-micelle approach for in vivo biological sensing applications.

Photophysical properties of ß-substituted free-base corroles.

Corroles are an emergent class of fluorophores that are finding an application and reaction chemistry to rival their porphyrin analogues. Despite a growing interest in the synthesis, reactivity, and functionalization of these macrocycles, their excited-state chemistry remains undeveloped. A systematic study of the photophysical properties of ß-substituted corroles was performed on a series of free-base ß-brominated derivatives as well as a ß-linked corrole dimer. The singlet and triplet electronic states of these compounds were examined with steady-state and time-resolved spectroscopic methods, which are complemented with density functional theory (DFT) and time-dependent DFT calculations to gain insight into the nature of the electronic structure. Selective bromination of a single molecular edge manifests in a splitting of the Soret band into x and y polarizations, which is a consequence of asymmetry of the molecular axes. A pronounced heavy atom effect is the primary determinant of the photophysical properties of these free-base corroles; bromination decreases the fluorescence quantum yield (from 15% to 0.47%) and lifetime (from 4 ns to 80 ps) by promoting enhanced intersystem crossing, as evidenced by a dramatic increase in knr with bromine substitution. The nonbrominated dimer exhibits absorption and emission features comparable to those of the tetrabrominated derivative, suggesting that oligomerization provides a means of red-shifting the spectral properties akin to bromination but without decreasing the fluorescence quantum yield.

Metabolic tumor profiling with pH, oxygen, and glucose chemosensors on a quantum dot scaffold.

Acidity, hypoxia, and glucose levels characterize the tumor microenvironment rendering pH, pO2, and pGlucose, respectively, important indicators of tumor health. To this end, understanding how these parameters change can be a powerful tool for the development of novel and effective therapeutics. We have designed optical chemosensors that feature a quantum dot and an analyte-responsive dye. These noninvasive chemosensors permit pH, oxygen, and glucose to be monitored dynamically within the tumor microenvironment by using multiphoton imaging.

Two-photon oxygen sensing with quantum dot-porphyrin conjugates.

Supramolecular assemblies of a quantum dot (QD) associated to palladium(II) porphyrins have been developed to detect oxygen (pO2) in organic solvents. Palladium porphyrins are sensitive in the 0-160 Torr range, making them ideal phosphors for in vivo biological oxygen quantification. Porphyrins with meso pyridyl substituents bind to the surface of the QD to produce self-assembled nanosensors. Appreciable overlap between QD emission and porphyrin absorption features results in efficient Förster resonance energy transfer (FRET) for signal transduction in these sensors. The QD serves as a photon antenna, enhancing porphyrin emission under both one- and two-photon excitation, demonstrating that QD-palladium porphyrin conjugates may be used for oxygen sensing over physiological oxygen ranges.

Porphyrin complexes of the period 6 main group and late transition metals.

Metalloporphyrin complexes of the period six metals gold, mercury, thallium, lead and bismuth are often overlooked in favour of their lighter congeners. These complexes exhibit unusual coordination geometries, prominently featuring the metal centre residing out the porphyrin plane. Not only are these compounds chemically interesting, but several applications for these complexes are beginning to emerge. Gold and bismuth porphyrins have medicinal applications including novel chemotherapeutics and sensitizers for α-radiotherapy, while gold porphyrins have applications in materials chemistry and catalysis. This perspective serves to highlight trends in the synthesis and structure of these heavy metal complexes as well as illustrate the considerations necessary for rationally designing elaborate porphyrin architectures.