All plants, animals, and many microorganisms take advantage of the instability of oxygen to power life processes. The molecules in the food are oxidized, and the energy is used to build new molecules, swim or crawl, and reproduce. However, food is not oxidized by burning in our body. It is oxidized in a number of steps, each of which is carefully controlled and designed to capture as much available energy as possible.
Cytochrome c oxidase controls the final step of food oxidation. At this point, the atom itself has been completely removed, leaving only a few electrons from the food molecule. The cytochrome c oxidase shown here acquires these electrons and attaches them to the oxygen molecule. Then, some hydrogen ions are added as well, forming two water molecules.
Cytochrome c oxidase (CcO) is the terminal enzyme of the mitochondrial respiratory chain and is also used for red and near-infrared light therapy (PBM).The core target. This non-invasive therapy utilizes red light (RL) at 620–700 nm and near-infrared light (NIR) at 700–1440 nm to regulate cellular metabolism and is widely used in areas such as wound healing, anti-inflammatory, and nerve repair.
1. CCO: The "Gatekeeper" of Cellular Energy Conversion
CCO is a key protein complex (molecular weight approximately 200 kDa) on the inner mitochondrial membrane and consists of 13 subunits with an active center containing heme a and a copper ion center (CuB). During cellular respiration, it catalyzes two key processes:
Electron transport: the redox change of iron atoms in heme, which transfers electrons to oxygen molecules and reduces them to water;
Proton pumping: Protons(H⁺) in the matrix are pumped into the mitochondrial membrane space to establish a transmembrane electrochemical gradient.
4 Fe² + 8 H+ + O₂→ 4 Fe3+ + 2 H2O + 4 H+
This total process is central to the third stage of aerobic respiration. For every 4 electrons transferred, 1 O₂ molecule is reduced, and 4 protons are pumped out at the same time, driving ATP synthase to produce adenosine triphosphate (ATP). Approximately 90% of the energy produced by glucose via the glycolytic-tricarboxylic acid cycle pathway is dependent on the proton gradient established by the CcO.
2. Photobiological properties of CcO: how red light "activates" mitochondria
(1) Proton pump mechanism of CcO
CCO drives proton pumping, which is one of the most subtle functions of Cytochrome c Oxidase (CcO), the essence of which is to convert the energy released by electron transfer into a transmembrane proton gradient, thereby driving ATP synthesis.
In the active center of the CcO, copper ions (CuB) and iron ions (heme a3-Fe) together form a dinuclear center, which synergistically undergoes valence changes during the oxygen reduction process, driven by electron transport and oxygen reduction processes.
Heme a₃-Fe Fe²⁺↔Fe³⁺↔Fe4+
controls the opening and closing of the D channel (inhalation of protons)
Cuʙ Cu⁺↔Cu²⁺
triggers Glu²⁸⁶ energy storage/release (pumping out protons)
The role of key molecular structures
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Structure |
function |
Red light regulates the target
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D channel |
The path of protons entering from the matrix
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Conformational flexibility enhancement
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Glu²⁸⁶
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"Molecular springs" that store energy
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Vibration energy increases
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Heme a₃-Cuʙ
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Oxygen binding and reduction sites
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NO dissociation
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Tyr244
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An intermediate that delivers protons to oxygen
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Redox accelerates
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(2) CcO absorbs photons to initiate an electron transition
The heme a and copper centers of CcO are the main absorbers of red and near-infrared light. When the energy of a photon matches the difference between its electron energy level, the electron transitions to the excited state, initiating the following chain reactions that triggers NO dissociation.
◦ The active center of the ground-state CcO is inhibited by nitric oxide (NO) binding (≈ 60% of enzyme activity is blocked)
◦ Red photons are absorbed by heme a → Electrons transition from ground state (S₀) to excited state (S₁)
◦ The energies of the excited state weaken the Fe²⁺-NO bond energy and dissociate the NO (Lane, 2006)
◦ Enzyme activity is restored, and the rate of oxygen metabolism is increased
Energy Conversion Path:
Photon energy → Electron excitation (femtosecond grade) → Vibrational relaxation (picosecond grade) → Conformational change (millisecond order) → Proton pumping enhancement
Essence: Light energy is converted into intramolecular energy (vibration/rotation), which in turn drives biological functions.
Red light accelerates this cycle by reducing the electron transport energy barrier and improves the efficiency of proton gradient establishment.
Accelerated Electron Injection
• Red light (620–850 nm) is absorbed by the heme a and Cuʙ centers of the CCO
• Reduced electron transport barrier, making it easier for electrons to flow from cytochrome c to Cuʙ/heme a₃
• Results: Fe³⁺→ Fe²⁺reduction rate increased by 2–3 times
Cu²⁺→ Cu⁺ reduction rate increased by 1.8–2.5 times
Experimental evidence: The electron transfer rate of CcO increased from 220 e⁻/s to 480 e⁻/s after 808 nm laser irradiation (Karu, Photomed. Laser Surg. 2010)
Removal of NO inhibition (indirect enhancement)
• Blocking effect of NO:
NO binds to Fe²⁺→ blocking O₂ binding
NO binds to Cu⁺ → inhibiting electron transport
• The role of red light:
Photon energy (1.5–2.0 eV) → weakens the metal-NO bond → dissociation
Results: Fe²⁺restored O₂binding energy Cu⁺restored electron transport function
Data: 660 nm light irradiation increased the NO dissociation rate.（Cheng K et.al, 2021）
The key parameter window effect : The effect of PBM is biphasic (Arndt-Schulz curve). Low doses (energy density 1–5 J/cm²) stimulate cell activity, while high doses (>100 J/cm²) inhibit function. For example, at wavelengths of 660 nm and 810 nm, CcO absorbs photons most efficiently.
summary
1. Biological role of CcO
As a terminal enzyme in the mitochondrial respiratory chain, it catalyzes the reduction of oxygen molecules to water (4e⁻+ 4H⁺+ O₂→ 2H2O), while pumping protons to establish a transmembrane gradient that drives 90% ATP synthesis.
2. The path of the red light to activate the CcO
Photon absorption: Heme a/ CuB absorbs red light→ electron transition→ weakens Fe²⁺-NO bond→ relieves NO inhibition.
Enzyme activity recovery: Accelerated electron transport (2–3-fold increase in rate) and proton pumping to enhance ATP production.
3. Difference in intensity
Optimum wavelengths: 660 nm (red) and 810 nm (near-infrared). Optimum energy density: 1–5 J/cm² (low-dose activation, high-dose inhibition)
Key references:
1. Karu, T. (1999). Primary and secondary mechanisms of action of visible to near-IR radiation on cells. Journal of Photochemistry and Photobiology B: Biology, 49(1), 1–17.
2. Poyton RO, Ball KA. Therapeutic photobiomodulation: nitric oxide and a novel function of mitochondrial cytochrome c oxidase. Discov Med. 2011 Feb;11(57):154-9. PMID: 21356170.
3.Karu, T. I. (2010). Mitochondrial signaling in mammalian cells activated by red and near-IR radiation. Photomedicine and Laser Surgery, 28(2), 159–160.
4.Wong-Riley, M.T. et al. (2005). Photobiomodulation directly benefits primary neurons functionally inactivated by toxins. Journal of Biological Chemistry, 280(6), 4761–4771.
5. Lane, N. (2006). Cell biology: Power games. Nature, 443(7114), 901–903.
6. Cheng K, Martin LF, Slepian MJ, Patwardhan AM, Ibrahim MM. Mechanisms and Pathways of Pain Photobiomodulation: A Narrative Review. J Pain. 2021 Jul;22(7):763-777. doi: 10.1016/j.jpain.2021.02.005. Epub 2021 Feb 23. PMID: 33636371; PMCID: PMC8277709.
