Introduction

Heme is an iron-containing porphyrin compound that functions as a prosthetic group for a variety of proteins, including myoglobin, cytochromes, catalase, and nitric oxide synthase1,2,3. At the core of the heme molecule lies an iron atom capable of binding with small molecules such as oxygen and carbon monoxide4. Consequently, heme plays a pivotal role in oxygen transport and storage within living organisms5,6,7. As a crucial biomolecule, heme possesses the ability to catalyze a variety of chemical reactions due to its central iron atom8,9. Within the cytochrome P450 enzyme system, heme acts as the catalytic center, facilitating the oxidation of alkanes and alcohols10,11. For instance, it can convert alkanes into alcohols or oxidize alcohols into aldehydes or ketones. Chlorophenols, on the other hand, are a class of phenolic compounds containing chlorine atoms, widely used in industry as biocides, preservatives, disinfectants, and wood preservatives12,13. The diversity of chlorophenols in water is extensive, with the most common being pentachlorophenol (PCP), 2,4-dichlorophenol (2,4-DCP), and 2,4,6-trichlorophenol (2,4,6-TCP)14,15.

Chlorophenols are hazardous organic compounds that, when ingested over extended periods, can lead to liver and kidney dysfunction, neurological damage, and increased cancer risk16,17,18,19. Additionally, they pose a detrimental impact on aquatic ecosystems, affecting biodiversity, compromising water quality, and posing a potential threat to human health20,21. Given their potent toxicity, propensity to damage liver and kidneys, neurotoxicity, high carcinogenic risk, water pollution, and ecological threats, the degradation of chlorophenols in water is of paramount importance.

The degradation of chlorophenols requires specific treatment technologies such as advanced oxidation processes (AOPs) and biodegradation, which are often costly and operationally complex22,23. The detection of chlorophenols necessitates highly sensitive analytical methods, and their presence in water alongside other organic compounds complicates detection and monitoring15,24. Consequently, the direct experimental investigation of chlorophenol degradation in water is exceedingly challenging. Utilizing established theoretical techniques for exploration could potentially provide significant insights into the degradation of chlorophenols in water.

As heme can catalytically activate chlorophenols containing phenolic hydroxyl groups, it might facilitate the degradation of chlorophenols in water, thereby providing significant guidance for the management of chlorophenol pollution. Presently, with the rapid advancement of quantum computing technology, the computational simulations using density functional theory (DFT) and the B3LYP method have achieved a high degree of consistency with experimental values25,26,27,28,29. By employing the DFT/B3LYP method, the study of heme’s catalytic effects on common chlorophenols in water namely, 2,4-DCP, 2,4,6-TCP, and PCP can offer crucial data support for the application of heme in the treatment of chlorophenol pollution in water. For example, In the catalytic interaction between heme and chlorophenols, the observed changes in the spectroscopic properties of the resulting complexes may serve as valuable technical support, aiding our comprehension of heme’s role in the degradation of diverse chlorophenols, from pre-degradation through to post-degradation stages. Additionally, the nuanced variations in heme’s binding efficacy with various chlorophenols, in response to the fluctuating concentrations of these compounds in water, could provide further insights into the potential efficiency of heme as a treatment agent for water contaminated with chlorophenols.

Research methods and content

This study utilizes the Gaussian 16 quantum computational chemistry software30, employing density functional theory (DFT) and the B3LYP method31,32,33,34. Atomic bases of 6–311 g* are utilized for elements such as C, O, N, Cl, and H, while the LanL2MB basis set is applied for the Fe atom35,36. The investigation examines the structural parameter changes, Wiberg Bond Indices variations, natural charge distribution alterations, as well as the changes in infrared vibrational spectra, ultraviolet-visible absorption spectra, and molecular fluorescence spectra of 2,4-DCP, 2,4,6-TCP, and PCP before and after their complexation with heme. Initially, simulations of heme forming complexes with each of the three chlorophenols are conducted, and the presence of imaginary frequencies in the vibrational spectra is checked to verify the stability of these complexes. Upon confirming the stability of the complexes, further analysis is performed on their structural parameters, Wiberg bond orders, ultraviolet-visible absorption spectra, molecular fluorescence spectra, binding energies, and frontier molecular orbital energy levels.

Results and analysis

Bond length

The bond length refers to the average or equilibrium distance between two atoms within a molecule, measured when the molecule is in its ground state—the state of lowest molecular energy and greatest stability. This length is typically determined under the condition where the molecule adopts its lowest energy configuration37,38. The size of the bond length depends on the nature of the connected atoms and the type of chemical bond between them (such as single, double, or triple bonds). Within the same molecule, if the bond length between two atoms increases, it may indicate a weakening of the bond, as longer bonds generally correspond to lower electron density and lower bond energy39. Conversely, if the bond length decreases, it may suggest a strengthening of the bond, as shorter bonds typically signify higher electron density and higher bond energy40,41.

Table 1 displays the primary interatomic bond lengths of 2,4-DCP, 2,4,6-TCP, and PCP before and after their complexation with heme to form Heme-2,4-DCP, Heme-2,4,6-TCP, and Heme-PCP, respectively. The atomic numbering for these bond lengths is depicted in Fig. 1. The optimized structural diagrams of the complexes formed by the three chlorophenols with heme are shown in Fig. 2. It is evident that upon binding with heme, the C-C bond lengths in the aromatic ring and the C-Cl bond lengths in the chlorophenols are significantly extended. This bond length extension is likely linked to reduced electron delocalization within the aromatic ring, which may weaken the pi-electron cloud and subsequently diminish the overall structural stability. This indicates that the stability of the chlorophenol aromatic structure is compromised after binding with heme. This finding provides a reference for the activation and degradation of chlorophenols, potentially reducing their persistent toxicity in the environment.

Fig. 1
figure 1

Molecular Structures and Atomic Numbering of 2,4-Dichlorophenol (2,4-DCP), 2,4,6-Trichlorophenol (2,4,6-TCP), and Pentachlorophenol (PCP).

Full size image
Fig. 2
figure 2

Structural Diagrams of the Complexes Formed Between Heme and the Three Chlorophenols (Heme-2,4-DCP, Heme-2,4,6-TCP, Heme-PCP).

Full size image
Table 1 The main bond lengths (nm) of the three chlorophenols and their complexes with heme before and after formation.
Full size table

Wiberg bond indices (WBIs)

The WBIs is a measure that quantifies the distribution of bonding electrons across molecular orbitals, offering a glimpse into the electron density shared between two atoms42,43,44,45. It ranges from 0 to 1 for single bonds, values above 1 are indeed indicative of multiple bonds, such as double or triple bonds. WBIs serve as a numerical measure of the bond strength between atoms within a molecule. These indices are rooted in the natural orbital theory of quantum chemistry and offer a perspective on the degree of overlap among van der Waals orbitals. WBIs are themselves a dimensionless numerical measure used to quantify the bond order between two atoms within a molecule. A higher WBIs generally implies a stronger bond. While bond length provides insight into a bond’s physical nature, the WBIs is a more nuanced indicator of its electronic structure. Consequently, a reduction in the WBI value associated with a chemical bond within a molecule suggests that the bond may have been activated due to specific factors. Following the binding of chlorophenol with heme, variations in WBIs values can pinpoint certain activation sites on the chlorophenol molecule.

The primary WBIs for 2,4-DCP, 2,4,6-TCP, and PCP are presented before and after their complexation with heme to form Heme-2,4-DCP, Heme-2,4,6-TCP, and Heme-PCP, respectively, in Table 2 The data suggest that although the bond order values for the aromatic ring remain above 1 following heme binding, indicating a persistence of multiple bonding tendencies between adjacent atoms in the ring. However, the bond orders of the C-Cl atoms in each chlorophenol have decreased from values above 1 to below 1 upon complex formation, signifying a marked weakening of the bond between the Cl atom and the C atom in the aromatic ring, from a multiple bond to a single bond. This clearly illustrates that heme binding to chlorophenols activates the chlorine atoms on the aromatic ring, making them more susceptible to detachment from the ring46.

Table 2 The WBIs values of the aromatic ring in three chlorophenols and their corresponding complexes with Heme (heme) before and after complexation.
Full size table

Infrared vibration spectroscopy

Infrared spectroscopy (IR) is a powerful tool for investigating molecular structure and the characteristics of chemical bonds. It achieves this by detecting the absorption of light in the infrared region, which corresponds to the vibrational modes of molecules47,48. Figure 3 presents the infrared vibration spectra for the three chlorophenols (2,4-DCP, 2,4,6-TCP, and PCP), while Fig. 4 shows the spectra for the complexes formed when these chlorophenols bond with heme.

In IR spectroscopy, the appearance of imaginary frequencies can indicate an unstable or transition state molecular structure, as these frequencies correspond to non-stable equilibrium points in molecular vibrations. However, both Figs. 3 and 4 lack any imaginary frequencies in all vibrations, indicating that the molecular structures under examination—the three chlorophenols and their complexes with heme—are stable in their ground state. This is because stable molecules exhibit vibrational modes that correspond to real frequencies, rather than imaginary ones.

IR spectra typically reveal the vibrational modes of various chemical bonds within a molecule, including stretching and bending vibrations. By examining the positions (wavenumbers), intensities, and shapes of these vibrational modes, one can deduce the presence of functional groups and the types of chemical bonds. For instance, characteristic absorption peaks corresponding to functional groups such as hydroxyl (-OH), amino (-NH), and carbonyl (C = O) can be observed in the IR spectra. Within the chlorophenols, absorption peaks for the hydroxyl group are present, whereas in heme, absorption peaks for both the carbonyl and hydroxyl groups are evident. Analysis of the infrared spectra in Figs. 3 and 4 allows us to conclude that the three chlorophenols and their complexes with heme are stable in their ground state. The spectral data can be further analyzed to explore their interactions and structural attributes.

Fig. 3
figure 3

Infrared spectra of 2,4-DCP, 2,4,6-TCP, PCP, and Heme.

Full size image
Fig. 4
figure 4

Infrared spectra of Heme-2,4-DCP, Heme-2,4,6-TCP, and Heme-PCP.

Full size image

Ultraviolet-visible (UV-Vis) absorption spectroscopy

UV-Vis absorption spectroscopy is a spectroscopic technique extensively utilized due to its reliance on the absorption properties of substances when exposed to electromagnetic radiation within the ultraviolet and visible light spectrums49,50. This method finds its way into various fields including chemistry, biochemistry, environmental science, and materials science, where it is pivotal for both quantitative measurements and qualitative assessments. UV-Vis spectroscopy is also instrumental in the identification and characterization of different compounds, as each exhibits a unique absorption spectrum. The distinct features of these spectra, such as peak positions, shapes, and intensities, serve to differentiate between various compounds. Comparative analysis with standard spectral data for known compounds allows for the qualitative analysis of unknown samples.

As Fig. 5 elucidates, three types of chlorophenols and heme display substantial UV absorption characteristics, with heme showcasing an exceptionally strong absorption profile. This anomaly can be attributed to the substantial π-π transitions occurring within heme, compounded by the molecule’s overall rigid, planar structure. The three chlorophenols present a comparable level of UV absorption without significant variation in absorption intensity, with absorption peaks occurring at 247 nm, 253 nm, and 260 nm, respectively. The absorption wavelengths exhibit a slight upward trend with the addition of chlorine atoms, which is a consequence of the electron-withdrawing nature of chlorine. The progressive increase in absorption wavelengths with more chlorine atoms in chlorinated phenols suggests a shift in the electronic structure of these compounds. This shift can have several implications for the degradation kinetics of more heavily chlorinated phenols: (i). Reactivity: More heavily chlorinated phenols may exhibit different reactivities compared to their less chlorinated counterparts. The presence of additional chlorine atoms can increase the electron-withdrawing effect, making the molecule more reactive towards certain oxidative degradation pathways. This could potentially accelerate the degradation process under appropriate conditions. (ii). Selectivity of Degradation Processes: The increased absorption wavelengths might indicate changes in the energy levels of the molecular orbitals, which could influence the selectivity of degradation processes. Certain oxidative agents or catalysts may preferentially target more heavily chlorinated phenols due to their unique electronic configurations. (iii). Intermediate Formation: Degradation of chlorinated phenols often involves the formation of intermediates. The presence of more chlorine atoms could lead to the formation of different or more complex intermediates, which might have different stabilities and reactivities. This could affect the overall degradation pathway and kinetics.

The formation of complexes between heme and the chlorophenols leads to notable alterations in the absorption wavelengths and intensities of the resulting complexes. Notably, Heme-2,4-DCP demonstrates a substantially higher absorption intensity compared to 2,4-DCP, with an absorption peak at 363 nm in the UV region. In contrast, the optimal absorption peaks of Heme-2,4,6-TCP and Heme-PCP are significantly attenuated when compared to Heme-2,4-DCP, and they fail to present clear, distinctive absorption peaks, with their absorption maxima occurring around 402 nm and 519 nm, respectively.

Examining the structures of the three complexes in Fig. 2, it becomes apparent that in the Heme-2,4-DCP complex, the angle between the heme molecule’s plane and that of the 2,4-DCP is the smallest, approaching a parallel configuration, which results in a heightened UV absorption intensity and clear absorption characteristics. In the cases of the Heme-2,4,6-TCP and Heme-PCP complexes, an increase in the number of chlorine atoms contributes to a larger angle between the chlorophenol and the heme’s planes, leading to a considerable redshift in absorption wavelengths, as observed in the UV-visible absorption spectral characteristics detailed in Fig. 5. This phenomenon could be attributed to the escalation in the count of electron-withdrawing Cl atoms, which perturbs the electronic energy levels within the molecule, thereby inducing a red shift in the absorption peak.

Fig. 5
figure 5

UV-Vis absorption spectra of three chlorophenols, hemin, and the complexes formed by the interaction of three chlorophenols with hemin.

Full size image

Molecular fluorescence spectroscopy

Molecular fluorescence spectroscopy capitalizes on the inherent property of molecules to absorb light at specific wavelengths and undergo a series of energy transformation processes ( such as internal conversion, vibrational relaxation, and intersystem crossing ), ultimately emitting light of longer wavelengths. This emitted light, known as fluorescence, typically resides within the visible or near-ultraviolet spectrum. This emitted light, known as fluorescence, typically falls within the visible or near-ultraviolet spectrum. This spectroscopic technique is invaluable for both qualitative and quantitative analyses of substances51,52,53. By comparing the fluorescence profiles of unknown samples against those of established standard substances, it is possible to identify and characterize the compounds present54. Figure 6 illustrates the fluorescence spectra of three chlorophenols, heme, and the complexes formed by the interaction of heme with these chlorophenols. The fluorescence peaks for Heme-2,4-DCP and Heme-2,4,6-TCP are particularly pronounced, rendering the fluorescence signals from other molecules in the figure relatively indistinct. The robust fluorescence observed for Heme-2,4-DCP can be attributed to its extensive conjugated molecular system, which features a rigid, planar structure. As Fig. 2 demonstrates, Heme-2,4,6-TCP exhibits an even greater degree of planarity than Heme-2,4-DCP, and the addition of an extra chlorine atom contributes to a reduction in fluorescence intensity and a slight shift towards longer wavelengths for Heme-2,4,6-TCP. The underlying causes of this phenomenon can be summarized as follows: firstly, a decrease in the electron cloud density within the chromophore—the segment of the molecule that facilitates fluorescence emission—reduces the likelihood of electronic transitions from the ground state to the excited state, consequently attenuating the fluorescence intensity. Secondly, an augmentation in electron-withdrawing groups could potentially depress the molecule’s excited state energy level, prompting the absorption peak to migrate toward the longer wavelength spectrum (the red end), thus triggering a red shift. Furthermore, this reduction in the excited state energy level might also render the excited state increasingly susceptible to decay via non-radiative channels, ultimately diminishing the fluorescence intensity. This fluorescence behavior serves as a distinctive identifier for the complexes formed between heme and the three chlorophenols, offering crucial diagnostic information for the qualitative and quantitative analysis of heme in the presence of 2,4-DCP and 2,4,6-TCP.

Fig. 6
figure 6

Fluorescence spectra of the three chlorophenols, heme, and the complexes formed between the three chlorophenols and heme.

Full size image

Binding energy

Intermolecular binding energy encompasses the energy associated with the forces that act between molecules, including van der Waals forces, hydrogen bonds, ionic bonds, covalent bonds, and others55,56. This energy is fundamental to understanding the physicochemical properties of substances, the molecular structure, intermolecular interactions, and the kinetics of chemical reactions. The difference in binding energy between reactant and product molecules can be a determining factor in the driving force of a reaction and the heat effect observed.

The intermolecular binding energy is particularly critical in the interaction between the catalyst matrix molecule and the receptor molecule, which directly impacts the selectivity of the catalyst for the receptor. Table 3 provides the binding energies for the complexes formed between heme and 2,4-dichlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol. It is evident that in the Heme-PCP complex, the binding energy between heme and PCP is the most substantial. This can be attributed to the increase in the number of chlorine atoms in the chlorophenol, which leads to a larger angle between the chlorophenol and heme in Heme-PCP, nearly achieving a perpendicular configuration. This configuration minimizes molecular repulsion between the heme and PCP planes, thereby enhancing the binding force significantly.

Similarly, the increased molecular binding energy in Heme-2,4,6-TCP compared to Heme-2,4-DCP can be explained by the same principle. The data in Table 3 also suggests that heme is most responsive to the selectivity of PCP, demonstrating a strong binding ability or an efficient capacity to capture PCP.

Table 3 Binding energy of each chlorophenol to Heme (kJ/mol).
Full size table

Frontier molecular orbitals (FMOs)

Frontier Molecular Orbitals (FMOs) are the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) within a molecule57,58. These two orbitals are pivotal in determining the electronic structure and chemical reactivity of the molecule. The equation for calculating the energy gap is as follows:

$$\Delta {\text{E }} = {\text{ ELUMO }} - {\text{ EHOMO }}$$
(1)

In the Eq. (1), ΔE represents the energy gap value, while ELUMO and EHOMO denote the energy levels of the LUMO and the HOMO, respectively.

The size of the energy gap has significant implications for a molecule’s properties: (i) Chemical Reactivity: Molecules with a smaller energy gap are more chemically reactive since electrons can transition more readily from the HOMO to the LUMO, participating in chemical reactions. In contrast, molecules with a larger energy gap are less reactive. (ii) Stability: Molecules with a larger energy gap tend to be more stable because they are less susceptible to oxidation (loss of electrons) or reduction (gain of electrons). (iii) Supramolecular Chemistry and Self-Assembly: The energy gap can affect intermolecular interactions, such as the formation of electron donor-acceptor complexes.

Table 4 presents the frontier molecular orbital energy values and energy gaps for the complexes formed between heme and different chlorophenols. It is clear that the energy gap for Heme-PCP is substantially lower than those for Heme-2,4-DCP and Heme-2,4,6-TCP, suggesting that Heme-PCP is the most activated complex. This is in line with the strongest binding between heme and PCP, as indicated by the molecular binding energy, resulting in the highest activation of PCP by heme. When combined with Table 3, which shows that heme is most sensitive to PCP and has the highest capture capability, these findings provide crucial reference information for understanding the activation and degradation of chlorophenols.

Table 4 The frontier molecular orbital energy ( EHOMO and ELUMO ) and energy gaps ( EL−H ) value ( eV ) of the Heme-XCP complexes.
Full size table

Conclusion

In examining the activation of 2,4-DCP, 2,4,6-TCP, and PCP using heme, it is apparent from the alterations in molecular structure and bond order that heme has exerted substantial activating effects on these chlorophenols. This validation underscores the capacity of heme to form stable complexes with 2,4-DCP, 2,4,6-TCP, and PCP, as evidenced by the distinct changes in ultraviolet absorption peak characteristics upon complex formation. Such changes can be leveraged for the detection and analysis of chlorophenols in water using heme as a probe. Within the complexes of heme and 2,4-DCP, 2,4,6-TCP, and PCP, the Heme-2,4-DCP and Heme-2,4,6-TCP display significant molecular fluorescence, an attribute not observed in 2,4-DCP and 2,4,6-TCP individually. This fluorescence property could significantly aid in the qualitative or quantitative detection of 2,4-DCP and 2,4,6-TCP via heme. More significantly, this could offer substantial evidence supporting the implementation of a real-time monitoring system for the heme-catalyzed reactions of diverse chlorophenols in practical scenarios. Although heme exhibits minimal fluorescence characteristics with PCP, the binding energy and the energy gaps of the frontier molecular orbitals suggest that heme has a stronger binding energy and a lower energy gap with PCP than with 2,4-DCP and 2,4,6-TCP, indicating a clear preference of heme for the activation of PCP.