Chemiexcitation: Mammalian Photochemistry in the Dark†

Abstract

Light is one way to excite an electron in biology. Another is chemiexcitation, birthing a reaction product in an electronically excited state rather than exciting from the ground state. Chemiexcited molecules, as in bioluminescence, can release more energy than ATP. Excited states also allow bond rearrangements forbidden in ground states. Molecules with low-lying unoccupied orbitals, abundant in biology, are particularly susceptible. In mammals, chemiexcitation was discovered to transfer energy from excited melanin, neurotransmitters, or hormones to DNA, creating the lethal and carcinogenic cyclobutane pyrimidine dimer. That process was initiated by nitric oxide and superoxide, radicals triggered by ultraviolet light or inflammation. Several poorly understood chronic diseases share two properties: inflammation generates those radicals across the tissue, and cells that die are those containing melanin or neuromelanin. Chemiexcitation may therefore be a pathogenic event in noise- and drug-induced deafness, Parkinson's disease, and Alzheimer's; it may prevent macular degeneration early in life but turn pathogenic later. Beneficial evolutionary selection for excitable biomolecules may thus have conferred an Achilles heel. This review of recent findings on chemiexcitation in mammalian cells also describes the underlying physics, biochemistry, and potential pathogenesis, with the goal of making this interdisciplinary phenomenon accessible to researchers within each field.

Figure 1.

Exciting electrons in a simple molecule. Left: Electron configuration in the outer molecular orbitals of the C=O of the simplest carbonyl compound, formaldehyde (H₂C=O). In the ground state S₀, paired electrons in an orbital have opposing spins per the Pauli exclusion principle (a “singlet state”). Exciting one electron to a higher-energy orbital can retain the opposing spins, giving singlet excited states S₁ or the higher energy S₂. This is a “spin allowed” process. If the excited electron’s spin flips, giving the former partners parallel spins, the excited state – the aggregate of all the orbitals – is a triplet state T₁ and this state has lower energy than S₁. Returning to the ground state S₀ requires the spin to spontaneously reverse (“flip”) and is “spin forbidden”; since triplet states cannot readily return to S₀, they have long lifetimes that allow a variety of harmless and harmful modes of dissipating the energy. σ and π are bonding orbitals, n is nonbonding, and σ* and π* are antibonding. Only the highest-energy of two n orbitals is shown (both are shown in Fig. 2). HOMO is the highest occupied molecular orbital, after filling with electrons from the lowest energy orbital up according to the Aufbau principle. LUMO is the lowest unoccupied molecular orbital, available for entry of an electron or electron pair. Right: The transition between orbitals for each state, with arrow length proportional to the energy required. Modified from (312, 313, 1).

Figure 2.

Orbital shapes of the ground state and excited states of a carbonyl. Left: Computed molecular orbitals of formaldehyde. Zero eV is the energy of an isolated electron in a vacuum; larger negative values reflect stronger bonding. Right: Enlargement showing ground-state electron configurations and frontier molecular orbitals of the C=O bond. Note the apparent 90° rotation between n and π* orbitals. Both electrons occupy both red and blue components of the wavefunction. Updated from (312, 313) with computations using Chemissian software.

Figure 3.

The available transitions between the ground state and electronically excited states (Jablonski diagram). A molecule in the ground state, usually the singlet state S₀, can be excited by absorbing a photon that excites an electron to one of the vibrational states of a higher electronic energy level such as S₁ or S₂. For O₂, the ground state is T₁. The vibrational energy quickly dissipates by collision (vibrational relaxation), leaving the molecule in the lowest vibrational state of its new electronic energy level. In S₂, this electronic energy can convert to an isoenergetic vibrational state of a lower electronic level S₁ (internal conversion) and again drop to the lowest vibrational level. In a flexible molecule, it can then return to the ground state by vibrational relaxation (gray energy levels); in a rigid molecule, it can instead emit a photon as fluorescence. It is generally “forbidden” (i.e. rare) for the excited electron to flip its spin to create a triplet state T₁ (intersystem crossing), or for a T state to cross to S. Exceptions occur if the two states involve different orbitals (the “El-Sayed rules”), e.g. singlet (π,π*) → triplet (n,π*), especially if the molecule contains a heavy atom such as Br or I (the “heavy atom effect” due to “spin-orbital coupling”) as described in the text. Because the return from T₁ to S₀ is forbidden, triplet states have long lifetimes that make them important for subsequent energy-transfer or chemical events. Triplet energy can be dissipated by slow emission of photons as phosphorescence. Jablonski energy levels reflect one geometry of a molecule. A different molecular geometry can give rise to unusual energy level regions in which the S₀ and T₁ state energies are close together or identical and allow a singlet ground state to cross to an excited triplet state without adding energy other than that which altered the ground state geometry; chemiexcitation operates in this way (see text). Adapted from (9, 313).

Figure 4.

Paths to an excited-state carbonyl. (a) The dioxetane, a strained four-atom ring usually found as a moiety on a larger molecule. It is unusual in being able to release large amounts of energy in a single reaction, creating long-lived, high-energy, electronically excited triplet states. Mild heat twists the two Os in opposite directions around the C–C bond (top), cleaving the O–O bond and then the C–C bond, leaving two carbonyls one of which becomes electronically excited. (b) A triplet-state carbonyl can be created by a reaction between two alkoxyl radicals or two alkylperoxyl radicals, which are produced by lipid oxidation or peroxidation, respectively. Electron transfer from a hydroquinone leaves a triplet carbonyl in the resulting semiquinone, a second transfer leaves a second excited carbonyl in the resulting quinone. When a dioxetanone or dioxetane cleaves, triplet carbonyls of higher energy arise. Firefly bioluminescence appears to rely on dioxetanone cleavage initiated by an enzyme. The mechanism is described in the text. Adapted from (16). (c) The apparent chemiexcitation reaction leading to a dioxetane on the eumelanin monomer DHICA, followed by cleavage to yield an excited triplet-state carbonyl (32, 1). UV radiation activates enzymes that synthesize the radicals ^•NO and O₂^•− for hours afterward. These radicals form peroxynitrite, ONOO⁻, which oxidizes eumelanin or its monomer DHICA (5,6- dihydroxyindole-2-carboxylic acid) and allows ambient O₂ to create a dioxetane on the melanin. Thermolytic cleavage of the dioxetane creates an electronically excited triplet state (*).

Figure 5.

Potential energy surfaces for dioxetane cleavage and CPD creation. (a) The energy contained in a dioxetane depends on the geometry of the O–O and C–C bonds. As heat-induced vibration twists the two Os apart at an increasingly large angle, the O–O bond stretches and the dioxetane’s ground-state energy S₀ increases until it matches that of an excited triplet state T₁, allowing easy intersystem crossing to T₁ at the diradicaloid transition state seam (TS). Further stretching lowers the ground-state energy, making reversal unlikely, and cleaves the C–C bond. The reaction product with two carbonyls is then born already in the excited state. The dark blue region on the right is the underside of the ground state surface which, on the left, is seen from the top. (b) The energy of a pair of adjacent pyrimidines in DNA also depends on their geometry. The energy required to reach the excited singlet state, as after UVC, depends on the degree to which heat-induced fluctuations twisted substituents around the 5=6 double bond or brought the two pyrimidines in close vertical and angular alignment (y and x axes). Torsion around the 5=6 double bond begins to resemble an excited diradicaloid state, in which the 5-6 is a single bond and the substituents and orbitals are freer to twist around the bond. Given sufficient twist and a close proximity of the two nucleobases, the energy approaches that of the excited state in the region near the conical intersection on the left. Small amounts of energy from a photon or triplet carbonyl then suffice to excite an electron from the S₀ ground state to an excited singlet state such as S₁. Conversely an excited molecule can rapidly dissipate energy and return to the ground state through the conical intersection, recreating the original two bases. With greater twist and closer proximity, however, the molecule finds itself near the conical intersection on the right. The pyrimidine pair forms 5-5 and 6-6 bonds joining them and, if the energy dissipates here, they drop to the ground state carrying the ring that makes a cyclo-butane pyrimidine dimer. Panel (a) from (68), copyright by the American Chemical Society (2013). Panel (b) courtesy of L. Blancafort, Univ. Girona.

Figure 6.

Where triplet-state energy goes. The energy of an excited triplet carbonyl, which has diradical character, is dissipated along both harmless and harmful pathways. Left to right: Energy is converted to heat (thermal deactivation) or to light emission (phosphorescence, Ph). A molecule in the triplet state can transfer its excited electron to another triplet state molecule, converting the latter to a singlet that generates fluorescence (triplet-triplet annihilation) (2). When a donor collides with an acceptor A, direct energy transfer can occur by Dexter electron exchange, with a high energy electron in the donor exchanging places with a lower energy electron in the acceptor. This is the likely mechanism for creating CPDs in the dark. Triplet acceptors that readily cross to the singlet state and fluoresce are useful for detecting poorly luminescing triplet states. Electron exchange with ambient molecular oxygen, which is a triplet in the ground state, creates singlet oxygen – an excited state of O₂ that is much more reactive because it can now react with the plethora of compounds that are singlets. If energy is transferred to a molecule having conjugated double bonds, such as sorbate or β-carotene, the diradical character of the newly excited double bond allows it to isomerize around the remaining single bond, dissipating the excess energy as heat by vibrational relaxation. This protection is the function of β-carotene and lycopene in the chloroplast’s reaction center. An excited carbonyl’s diradical character can also initiate [2+2] cycloaddition reactions (Paterno-Buchi reaction). Alternatively, it can facilitate hydrogen abstraction from an H donor (e.g., alcohols and 1,4-dienes), sometimes with C-C bond homolysis. Adapted from (16, 33).