Photoinduced Ligand Dissociation Research Articles

Reversible Photoinduced Ligand Substitution in a Luminescent Chromium(0) Complex.

Light-triggered dissociation of ligands forms the basis for many compounds of interest for photoactivated chemotherapy (PACT), in which medicinally active substances are released or "uncaged" from metal complexes upon illumination. Photoinduced ligand dissociation is usually irreversible, and many recent studies performed in the context of PACT focused on ruthenium(II) polypyridines and related heavy metal complexes. Herein, we report a first-row transition metal complex, in which photoinduced dissociation and spontaneous recoordination of a ligand unit occurs. Two scorpionate-type tridentate chelates provide an overall six-coordinate arylisocyanide environment for chromium(0). Photoexcitation causes decoordination of one of these six ligating units and coordination of a solvent molecule, at least in tetrahydrofuran and 1,4-dioxane solvents, but far less in toluene, and below detection limit in cyclohexane. Transient UV-vis absorption spectroscopy and quantum chemical simulations point to photoinduced ligand dissociation directly from an excited metal-to-ligand charge-transfer state. Owing to the tridentate chelate design and the substitution lability of the first-row transition metal, recoordination of the photodissociated arylisocyanide ligand unit can occur spontaneously on a millisecond time scale. This work provides insight into possible self-healing mechanisms counteracting unwanted photodegradation processes and seems furthermore relevant in the contexts of photoswitching and (photo)chemical information storage.

Ruthenium Pyrazole Complexes: A Family of Highly Active Metallodrugs for Photoactivated Chemotherapy.

Ruthenium complexes bearing bis pyrazole (pzH) ligands, cis-[Ru(bpy)2(R-pzH)2]2+ (bpy = 2,2'-bipyridine, R = -H, -Cl), were examined as photoactivated anticancer prodrugs. A dicationic pyrazole complex deprotonated to give monocationic pyrazole-pyrazolate complexes, cis-[Ru(bpy)2(R-pz-)(R-pzH)]+, in an aqueous solution with pKa values of 9.5 and 7.2 for R = H and R = Cl, respectively. Upon deprotonation, relative quantum yields of photosubstitution decreased while lipophilicity of the complexes increased according to the measurements of water-octanol coefficients. The ruthenium complex with 4-chloropyrazole ligands displayed high cytotoxicity upon light irradiation (IC50 = 0.060 ± 0.016 μM) toward lung cancer cells, which was 7 times higher than that in the dark (IC50 = 0.44 ± 0.07 μM). Additional experiments for the ruthenium R-pyrazole complexes indicated that (1) selective photodissociation of the 4-chloropyrazole ligand occurs from cis-[Ru(bpy)2(4-Clpz-)(4-ClpzH)]+, (2) photoinduced ligand dissociation is dominant rather than photoinduced generation of singlet oxygen (1O2), and (3) induction of cell death occurs via the intrinsic pathway of apoptosis.

Acetonitrile Ligand Photosubstitution in Ru(II) Complexes Directly from the 3MLCT State.

The excited states of the series [Ru(tpy)(L)(CH3CN)]n+ (1-11) (n = 1, 2) containing bidentate ligands L with varying electron-donating ability were characterized through Arrhenius analysis of the temperature dependence of their excited-state lifetimes. Complexes 1-11 undergo photoinduced CH3CN dissociation upon 450 nm irradiation with ligand exchange quantum yields that increase with the energy barrier to populating a dissociative triplet ligand field (3LF) state from the lowest-energy triplet metal-to-ligand charge transfer (3MLCT) excited state. Combined with DFT calculations, the data indicate that ligand photodissociation in 1-11 occurs directly from the 3MLCT state instead of a 3LF state. This finding is in contrast to the generally accepted mechanism for ligand photodissociation in Ru(II) complexes and indicates that alternative pathways for photoinduced ligand dissociation are available. These results can widely impact design principles for applications that require ligand photodissociation, such as photochemotherapy and photocatalysis, as well as for those where photosubstitution is undesirable, such as solar energy conversion.

Visible light-activated ruthenium-catalysed direct arylation at ambient temperature

We present a visible light-activated ruthenium-catalysed direct arylation of aryl CH bonds with aryl (pseudo)halides, which operates at ambient temperature with broad substrate scopes and excellent compatibility of functionalities. The key to success of this strategy is the efficient generation of active catalytic species via photo-induced ligand dissociation. The choices of the bases and solvents also have significant influence on the catalytic efficiency.

Converting an Almost Noncytotoxic Ru(II) Complex with Photolabile Ligands into a Highly Efficient PACT Agent

AbstractRu(II) complexes with weak ligand fields may undergo light‐induced ligand dissociation, and the resulted Ru(II) aqua complexes may bind with biomolecules such as DNA, showing potential as photoactivated chemotherapy (PACT) agents. However, Ru(II) complexes with efficient PACT activity are still rare. Some Ru(II) complexes exhibit efficient photoinduced ligand dissociation but poor cytotoxicity. It is speculated that the low nuclear accumulation levels may account for their low PACT efficacy. In order to confirm this hypothesis, the almost noncytotoxic [Ru(7‐OCH3‐dppz)(4‐OCH3‐py)4](PF6)2 (Ru1) is loaded on nucleus‐targeted C5N2 nanoparticles (NPs). Compared with the free Ru1, Ru1–C5N2 NPs exhibit significantly increased cellular uptake and nuclear accumulation. Therefore, Ru1–C5N2 NPs show efficient PACT activity toward various cancer cell lines (including cisplatin‐resistant one) with half maximal inhibitory concentration (IC50) values of 0.18 × 10−6–0.29 × 10−6 m and phototoxicity index (IC50dark/IC50light) values above 137 under both normoxic and hypoxic conditions. Moreover, Ru1–C5N2 NPs also exhibit efficient PACT activity toward cisplatin‐resistant 3D multicellular tumor spheroids upon two‐photon irradiation (800 nm). The same strategy is also feasible to greatly improve the PACT activity of [Ru(7‐OCH3‐dppz)(py)4]2+, which itself only has a medium effect. The results may provide new sights for developing efficient Ru(II) PACT agents.

The Development of Ru(II)-Based Photoactivated Chemotherapy Agents.

Photoactivated chemotherapy (PACT) is a novel cancer treatment method that has drawn increasing attention due to its high selectivity and low side effects by spatio-temporal control of irradiation. Compared with photodynamic therapy (PDT), oxygen-independent PACT is more suitable for treating hypoxic tumors. By finely tuning ligand structures and coordination configurations, many Ru(II) complexes can undergo photoinduced ligand dissociation, and the resulting Ru(II) aqua species and/or free ligands may have anticancer activity, showing their potential as PACT agents. In this mini-review, we summarized the progress in Ru(II)-based PACT agents, as well as challenges that researchers in this field still face.

A photoactivatable chemotherapeutic Ru(II) complex bearing bathocuproine ligand efficiently induces cell death in human malignant melanoma cells through a multi-mechanistic pathway

Photoactivated chemotherapy (PACT) is an emerging strategy for targeted cancer therapy. Strained Ru complexes with pseudo-octahedral geometry may undergo photo-induced ligand dissociation, forming aquated photoproducts that are significantly more cytotoxic compared to the precursor complex. The complexes investigated were the strained complex [Ru(bpy)2BC]Cl2 (where bpy = 2,2′-bipyridine and BC = bathocuproine) and its unstrained control [Ru(bpy)2phen]Cl2 (where phen = 1,10-phenanthroline). The uptake of [Ru(bpy)2BC]Cl2, assessed by ICP/MS, started immediately post-incubation and plateaued after 24 h. Active transport was found as the main mode of intracellular transport. Cell viability assays on A375 cells indicated a mean phototoxicity index of 340-fold, and the effect was shown to be primarily mediated by the aquated photoproducts rather than the dissociating ligands. A significant increase in ROS production and DNA damage was also observed. Flow cytometry confirmed the induction of early apoptosis at 48 h that proceeds to late apoptosis/necrosis by 72 h post-treatment. Western blot analysis of pro- and anti-apoptotic proteins revealed that apoptosis was mediated through an interplay between the intrinsic and extrinsic pathways, as well as autophagy and via inhibition of the MAPK and PI3K pathways. In conclusion, this study demonstrates that [Ru(bpy)2BC]Cl2 is a multi-mechanistic PACT drug which exhibits promising anticancer potential.

Pyrene modification enables enhanced two-photon excited ligand dissociation of a Ru(II) complex containing monodentate ligands

Ru(II) complexes which can undergo photo-induced ligand dissociation display potentials for biological applications in some aspects. However, their short excitation wavelengths limit tissue penetration and therefore applications. Two-photon technic is a general way to shift the excitation wavelength to the near-infrared region (NIR), but few Ru(II) complexes with photolabile ligands having large two-photon absorption cross sections (δ2) have been reported so far. In this work, by modification with a pyrene group on the tpy ligand, the resultant complex [Ru(tpy-py)(CH3CN)3]2+ (1) displayed enhanced two-photon induced ligand dissociation compared with the parent complex [Ru(tpy)(CH3CN)3]2+ (2), probably through an intramolecular energy transfer mechanism. The ligand dissociation process was investigated in detail by absorption, 1H NMR, ESI MS spectra and theoretical calculations.

A Ru(II)‐Based Nanoassembly Exhibiting Theranostic PACT Activity in NIR Region

AbstractPhotoactivated chemotherapy (PACT) has appealing merits over traditional chemotherapy as well as photodynamic therapy (PDT) by virtue of its spatial and temporal control on drug activity and oxygen‐independent mechanisms of action. However, the short photoactivation wavelengths, e.g., visible light–activated Ru(II)‐based PACT agents, limit the clinical application severely. In this work, a facile construction of supramolecular nanoparticles from a poly(ethylene glycol) (PEG)‐modified [Ru(dip)2(py‐SO3)]+ (abbreviated as Ru‐PEG, dip = 4,7‐diphenyl‐1,10‐phenanthroline, py‐SO3 = pyridine‐2‐sulfonate) and 1,3‐phenylenebis(pyren‐1‐ylmethanone) (BP) is shown. While Ru‐PEG may undergo photoinduced ligand dissociation and release anticancer species of [Ru(dip)2(H2O)2]2+, BP has extremely large two‐photon absorption cross sections (δ2) in the NIR region and intense fluorescence over the wavelengths where Ru‐PEG has strong absorption. Thus, two‐photon excitation of BP followed by an efficient Förster resonance energy transfer (FRET) from BP to Ru‐PEG may lead to a potent inactivation against cisplatin‐resistant cancer cells and 3D multicellular tumor spheroids (MCTSs). The residue fluorescence of BP also allows the cellular uptake of the particles to be visualized. This work provides a universal and convenient strategy to realize theranostic PACT in the ideal phototherapeutic window of 650–900 nm.

Photoinduced ligand dissociation follows reverse energy gap law: nitrile photodissociation from low energy 3MLCT excited states.

A series of Ru(ii)-terpyridine complexes containing electron-donating bidentate ligands are able to effectively photodissociate nitrile ligands using red light. A spectroscopic investigation of these complexes reveal that they follow anti-energy gap law behavior, providing further evidence that population of 3LF excited states is not necessary for photoinduced nitrile dissociation.

A nuclear permeable Ru(ii)-based photoactivated chemotherapeutic agent towards a series of cancer cells: in vitro and in vivo studies.

Ru(ii) polypyridyl complexes which can undergo photo-induced ligand dissociation and DNA covalent binding are considered as potential photoactivated chemotherapeutic (PACT) agents. Herein four pyridine-2-sulfonate (py-SO3-) ligand based Ru(ii) complexes [Ru(N-N)2(py-SO3)]+ (1-4) were synthesized and studied. All the complexes can undergo fast py-SO3- ligand dissociation and DNA covalent binding upon visible light irradiation. However, only complex 4 exhibited high photo-induced anticancer activities towards a series of cancer cells, with half maximal inhibitory concentration (IC50) values in 100-300 nM regions and phototoxicity index (PI) values of about 100. In particular, complex 4 can also kill cisplatin resistant SKOV-3 and A549 cancer cells with IC50 values in 200-400 nM regions and PI values of about 50, which should be the first report of Ru(ii) based PACT agents that are also effective towards cisplatin resistant cancer cells. Complex 4 exhibited much higher cell uptake and nuclear accumulation levels, which may be the main reasons for its high anticancer activities. The in vivo anticancer experiments indicated that complex 4 can inhibit tumor growth significantly with fewer side effects. Our results may provide guidelines for developing novel photoactivatable Ru(ii) anticancer agents.

New Ru(ii) photocages operative with near-IR light: new platform for drug delivery in the PDT window.

A series of Ru(ii) complexes bearing the tridentate 2,6-di(quinolin-2-yl)pyridine (dqpy) ligand were designed to undergo photoinduced ligand dissociation with red/near-IR light. The complexes [Ru(dqpy)(L)(CH3CN)]2+, where L = 2,2'-bipyridine (bpy, 1), 4,4'dimethyl-2,2'-bipyridine (Me2bpy, 2), and 1,10-phenanthroline (phen, 3). Complexes 1-3 exhibit red-shifted lowest energy metal-to-ligand charge transfer (MLCT) absorption maxima at ∼600 nm, as compared to the corresponding tpy (2,2';6',2''-terpyridine) complexes with MLCT bands at ∼565 nm which appear as shoulders to the MLCT bands at ∼455 nm. This shift is attributed to the lower energy LUMO afforded by the dqpy ligand when compared to tpy, as evidenced by the shift of the first reduction wave to ∼0.3 V more positive potentials in the former. In addition, the lowest MLCT maximum of [Ru(dqpy)(acac)(CH3CN)]+ (4; acac- = acetylacetonate) is observed at 770 nm, attributed to the additional increase in energy of the HOMO afforded by the presence of the π-donating acac- ligand and supported by calculations. Complexes 1-3 undergo ligand substitution upon irradiation with red light, λ irr ≥ 610 nm, and the ligand substitution photochemistry of 4 is accessible with near-IR light, λ irr ≥ 715 nm and λ irr = 735 nm. Complexes 1-4 exhibit similar quantum yields of ligand exchange, Φ L, with 450 and 600 nm irradiation, however, that of 4 is 2-3 times greater than those measured for 1-3. This enhancement is explained by the difference in ligand contributions to the HOMO. Density functional theory calculations predict partial dqpy ππ* character in the MLCT states of 1-3 and a mixed Ru/acac- → dqpy metal/ligand-to-ligand charge transfer (ML-LCT) state in 4. The photoreactivity of 1-4 with tissue-penetrating red and near-IR light, together with their exceptional dark stability (>48 h), makes the new Ru(ii)-dqpy platform ideal for the development of new complexes for photoinduced drug release and for other applications that require broad absorption from the ultraviolet and visible ranges into the near-IR, such as solar energy conversion.

DFT Investigation of Ligand Photodissociation in [RuII(tpy)(bpy)(py)]2+ and [RuII(tpy)(Me2bpy)(py)]2+ Complexes.

Photoinduced ligand dissociation of pyridine occurs much more readily in [Ru(tpy)(Me2bpy)(py)]2+ than in [Ru(tpy)(bpy)(py)]2+ (tpy = 2,2':6',2″-terpyridine; bpy = 2,2'-bipyridine, Me2bpy = 6,6'-dimethyl-2,2'-bipyridine; py = pyridine). The S0 ground state and the 3MLCT and 3MC excited states of these complexes have been studied using BP86 density functional theory with the SDD basis set and effective core potential on Ru and the 6-31G(d) basis set for the rest of the atoms. In both complexes, excitation by visible light and intersystem crossing leads to a 3MLCT state in which an electron from a Ru d orbital has been promoted to a π* orbital of terpyridine, followed by pyridine release after internal conversion to a dissociative 3MC state. Interaction between the methyl groups and the other ligands causes significantly more strain in [Ru(tpy)(Me2bpy)(py)]2+ than in [Ru(tpy)(bpy)(py)]2+, in both the S0 and 3MLCT states. Transition to the dissociative 3MC states releases this strain, resulting in lower barriers for ligand dissociation from [Ru(tpy)(Me2bpy)(py)]2+ than from [Ru(tpy)(bpy)(py)]2+. Analysis of the molecular orbitals along relaxed scans for stretching the Ru-N bonds reveals that ligand photodissociation is promoted by orbital mixing between the ligand π* orbital of tpy in the 3MLCT state and the dσ* orbitals that characterize the dissociative 3MC states. Good overlap and strong mixing occur when the Ru-N bond of the leaving ligand is perpendicular to the π* orbital of terpyridine, favoring the release of pyridine positioned in a cis fashion to the terpyridine ligand.

A ferrocenyl pyridine-based Ru(ii) arene complex capable of generating ·OH and 1O2 along with photoinduced ligand dissociation

A ferrocene modified Ru(ii) arene complex was developed to present dual activity of photoactivated chemotherapy (PACT) and photodynamic therapy (PDT).

New Ru(II) complexes for dual photoreactivity: ligand exchange and (1)O2 generation.

Uncovering the factors that govern the electronic structure of Ru(II)-polypyridyl complexes is critical in designing new compounds for desired photochemical reactions, and strategies to tune excited states for ligand dissociation and (1)O2 production are discussed herein. The generally accepted mechanism for photoinduced ligand dissociation proposes that population of the dissociative triplet ligand field ((3)LF) state proceeds through thermal population from the vibrationally cooled triplet metal-to-ligand charge transfer ((3)MLCT) state; however, temperature-dependent emission spectroscopy provides varied activation energies using the emission and ligand exchange quantum yields for [Ru(bpy)2(L)2](2+) (bpy = 2,2'-bipyridine; L = CH3CN or py). This suggests that population of the (3)LF state proceeds from the vibrationally excited (3)MLCT state. Because the quantum yield of ligand dissociation for nitriles is much more efficient than that for py, steric bulk was introduced into the ligand set to distort the pseudo-octahedral geometry and lower the energy of the (3)LF state. The py dissociation quantum yield with 500 nm irradiation in a series of [Ru(tpy)(NN)(py)](2+) complexes (tpy = 2,2':6',2″-terpyridine; NN = bpy, 6,6'-dimethyl-2,2'-bipyridine (Me2bpy), 2,2'-biquinoline (biq)) increases by 2-3 orders of magnitude with the sterically bulky Me2bpy and biq ligands relative to bpy. Ultrafast transient absorption spectroscopy reveals population of the (3)LF state within 3-7 ps when NN is bulky, and density functional theory calculations support stabilized (3)LF states. Dual activity via ligand dissociation and (1)O2 production can be achieved by careful selection of the ligand set to tune the excited-state dynamics. Incorporation of an extended π system in Ru(II) complexes such as [Ru(bpy)(dppn)(CH3CN)2](2+) (dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) and [Ru(tpy)(Me2dppn)(py)](2+) (Me2dppn = 3,6-dimethylbenzo[i]dipyrido[3,2-a:2',3'-c]phenazine) introduces low-lying, long-lived dppn/Me2dppn (3)ππ* excited states that generate (1)O2. Similar to [Ru(bpy)2(CH3CN)2](2+), photodissociation of CH3CN occurs upon irradiation of [Ru(bpy)(dppn)(CH3CN)2](2+), although with lower efficiency because of the presence of the (3)ππ* state. The steric bulk in [Ru(tpy)(Me2dppn)(py)](2+) is critical in facilitating the photoinduced py dissociation, as the analogous complex [Ru(tpy)(dppn)(py)](2+) produces (1)O2 with near-unit efficiency. The ability to tune the relative energies of the excited states provides a means to design potentially more active drugs for photochemotherapy because the photorelease of drugs can be coupled to the therapeutic action of reactive oxygen species, effecting cell death via two different mechanisms. The lessons learned about tuning of the excited-state properties can be applied to the use of Ru(II)-polypyridyl compounds in a variety of applications, such as solar energy conversion, sensors and switches, and molecular machines.

Steric and Electronic Factors Associated with the Photoinduced Ligand Exchange of Bidentate Ligands Coordinated to Ru(II).

In an effort to create a molecule that can absorb low energy visible or near-infrared light for photochemotherapy (PCT), the new complexes [Ru(biq)2 (dpb)](PF6 )2 (1, biq = 2,2'-biquinoline, dpb = 2,3-bis(2-pyridyl)benzoquinoxaline) and [(biq)2 Ru(dpb)Re(CO)3 Cl](PF6 )2 (2) were synthesized and characterized. Complexes 1 and 2 were compared to [Ru(bpy)2 (dpb)](PF6 )2 (3, bpy = 2,2'-bipyridine) and [Ru(biq)2 (phen)](PF6 )2 (4, phen = 1,10-phenanthroline). Distortions around the metal and biq ligands were used to explain the exchange of one biq ligand in 4 upon irradiation. Complex 1, however, undergoes photoinduced dissociation of the dpb ligand rather than biq under analogous experimental conditions. Complex 3 is not photoactive, providing evidence that the biq ligands are crucial for ligand photodissociation in 1. The crystal structures of 1 and 4 are compared to explain the difference in photochemistry between the complexes. Complex 2 absorbs lower energy light than 1, but is photochemically inert although its crystal structure displays significant distortions. These results indicate that both the excited state electronic structure and steric bulk play key roles in bidentate photoinduced ligand dissociation. The present work also shows that it is possible to stabilize sterically hindered Ru(II) complexes by the addition of another metal, a property that may be useful for other applications.

Isomerization initiated by photoinduced ligand dissociation in Ru(II) complexes with the ligand 2-p-tolylpyridinecarboxaldimine.

The excited state reactivity of Ru(ii) complexes continues to be a topic of intense investigation because of their widespread use in applications related to solar energy conversion, such as in photoswitches and for medicinal chemistry. In an effort to gain further understanding of photoinduced ligand exchange and isomerization in Ru(ii) complexes, various isomers of the formula [Ru(PTPI)2(CH3CN)2](2+) (PTPI = 2-p-tolylpyridinecarboxaldimine) were synthesized and characterized, as well as the tris-heteroleptic complexes cis-[Ru(bpy)(PTPI)(CH3CN)2](2+) (, bpy = 2,2'-bipyridine) and cis-[Ru(bpy)(PAP)(CH3CN)2](2+) (, PAP = 2-(phenylazo)pyridine). All of the complexes containing the PTPI ligand undergo photoinduced ligand exchange of the CH3CN ligands with the coordinating solvent. Each isolated di-substituted PTPI complex, however, also undergoes isomerization of the bidentate PTPI ligands upon irradiation in CH3CN to produce the same ratio of a mixture of 63% ε-[Ru(bpy)(PTPI)(CH3CN)2](2+) and 37% β-[Ru(bpy)(PTPI)(CH3CN)2](2+). Experiments reveal that the isomerization of PTPI only occurs after the process of ligand dissociation is initiated by light. Evidence of isomerization following photoinduced ligand dissociation was also observed in , but not for the analogous complex . Electronic structure calculations, which included the relative overall energies of the isomers, and emission data were used to explain the results. The work presented herein may be useful in the design of new complexes for solar energy conversion or photoswitching applications.

BODIPY-modified Ru(ii) arene complex—a new ligand dissociation mechanism and a novel strategy to red shift the photoactivation wavelength of anticancer metallodrugs

A Ru(II) arene complex [(η(6)-p-cymene)Ru(bpy)(py-BODIPY)](PF(6))(2), where bpy is 2,2'-bipyridine and py-BODIPY is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dye containing a pyridine group at the 8-position, was designed and synthesized. BODIPY modification renders the monodentate pyridine ligand with long wavelength absorbing capability, and an absorption maximum at 504 nm. Upon selective irradiation of the absorption band of the py-BODIPY ligand, the dissociation of the monodentate ligand occurs efficiently, followed by substitution by 9-ethylguanine if it is present in the solution. The photoinduced ligand dissociation quantum yield was measured to be 4.1% at 480 nm. The photoinduced electron transfer from the BODIPY chromophore to the Ru(II) arene moiety plays an important role in the ligand dissociation. Such a photosensitization strategy can be utilized to develop novel anticancer metallodrugs that may respond to light in the phototherapeutic window (650-900 nm).

Sol−Gel-Encapsulated Heme Proteins. Evidence for CO2 Adducts

Heme proteins have been the subjects of numerous studies of photoinduced ligand dissociation and recombination (e.g., O2, CO, and NO). Much of this work1 is aimed at unraveling the dynamical behavior of myoglobin (Mb), which lacks a crystallographic channel connecting the iron center with the solvent. Despite its importance as a conspicuous metabolic breakdown product, CO2 interactions with metalloproteins are not well understood. CO2 is required for the assembly of the active sites of some multinuclear metalloproteins (e.g., the Ni2 center of urease2). In carbonic anhydrase, zinc-coordinated hydroxide is believed3 to attack CO2, forming a bicarbonate product complex. Deoxyhemoglobin forms carbaminohemoglobin4 in red blood cells upon reaction of CO2 with the amino termini more readily than oxyhemoglobin does; oxygenation in the lungs causes CO2 release. More importantly, there is no direct evidence for the existence of a metal-ligated CO2 adduct of any metalloprotein. In addition to transporting gases, hemoglobin and myoglobin possess enzymatic activity: reactions of the met forms with peroxides generate intermediates5,6 containing ferryl (FeIVdO) and protein radical species that are capable of oxidizing small substrates. In the case of linoleic acid, it has been established7 that substrate oxidation by Mb occurs by direct ferryl oxygen atom transfer. In this paper, we demonstrate that ferrylMb oxidizes carbon monoxide to produce CO2. In addition, we present the first spectroscopic evidence for Fe-CO2 coordination in heme proteins. The reaction7 of metMb with excess hydrogen peroxide generates a metastable intermediate, containing ferryl iron, that decays within minutes to produce degraded forms of myoglobin. However, in a saturated CO solution, Mb is observed8 to produce a gaseous product. Table 1 presents the results of mass spectrometric analyses9 of the gases in sealed pear flasks containing combinations of metMb, H2O2, and CO in phosphate buffer (50 mM, pH 7.0). The m/e ) 44 signal clearly indicates that the product of the catalyzed reaction is CO2. However, the low solubility of CO2 precluded attempts to obtain the electronic spectrum of the product complex under these conditions. Following the suggestion of Ibers10 that liquid CO2 be used in searching for new CO2 complexes, we reasoned that supercritical CO2 would be an even better choice, owing to its gaslike viscosity. Furthermore, supercritical fluids constitute reaction media that are of emerging biotechnological interest.11