Temperature Derivative Spectroscopy Research Articles

Co and No Binding in Inducible Nitric Oxide Synthase

Nitric oxide synthases (NOSs) are homodimeric heme enzymes that catalyze the oxidative degradation of L-arginine (L-Arg) to nitric oxide (NO). Three structurally similar isoforms have been identified in endothelial cells (eNOS), neuronal tissues (nNOS) and in macrophages (iNOS). Different from eNOS and nNOS, iNOS is not present in resting cells but is expressed upon inflammatory and immunologic stimulation. As this isoform has been implicated in the pathogenesis of various diseases, there is a growing need for potent and highly selective inhibitors.The targeted development of potent inhibitors that are highly specific for iNOS requires detailed insights into the interaction between the ligand, the heme and the surrounding protein matrix on the molecular level. Therefore, we have investigated ligand and substrate binding in iNOSoxy using Fourier transform infrared (FTIR) spectroscopy in combination with temperature derivative spectroscopy (TDS). The physiological ligand O2 was replaced by carbon monoxide (CO) and NO. Both CO and NO allow us to exploit their excellent properties as spectroscopic probes. Previous studies on CO and NO migration in myoglobin have shown distinct differences in CO and NO migration despite their similar sizes.

Fourier transform infrared spectroscopy study ofligand photodissociation and migration in inducible nitric oxide synthase.

Inducible nitric oxide synthase (iNOS) is a homodimeric heme enzyme that catalyzes the formation of nitric oxide (NO) from dioxygen and L-arginine (L-Arg) in a two-step process. The produced NO can either diffuse out of the heme pocket into the surroundings or it can rebind to the heme iron and inhibit enzyme action. Here we have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy at cryogenic temperatures, using the carbon monoxide (CO) and NO stretching bands as local probes of the active site of iNOS. Characteristic changes were observed in the spectra of the heme-bound ligands upon binding of the cofactors. Unlike photolyzed CO, which becomes trapped in well-defined orientations, as indicated by sharp photoproduct bands, photoproduct bands of NO photodissociated from the ferric heme iron were not visible, indicating that NO does not reside in the protein interior in a well-defined location or orientation. This may be favorable for NO release from the enzyme during catalysis because it reduces self-inhibition. Moreover, we used temperature derivative spectroscopy (TDS) with FTIR monitoring to explore the dynamics of NO and carbon monoxide (CO) inside iNOS after photodissociation at cryogenic temperatures. Only a single kinetic photoproduct state was revealed, but no secondary docking sites as in hemoglobins. Interestingly, we observed that intense illumination of six-coordinate ferrous iNOS oxy-NO ruptures the bond between the heme iron and the proximal thiolate to yield five-coordinate ferric iNOS oxy-NO, demonstrating the strong trans effect of the heme-bound NO.

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase

Inducible nitric oxide synthase (iNOS) is a homodimeric heme enzyme that catalyzes the formation of nitric oxide (NO) from dioxygen and L-arginine (L-Arg) in a two-step process. The produced NO can either diffuse out of the heme pocket into the surroundings or it can rebind to the heme iron and inhibit enzyme action. Here we have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy at cryogenic temperatures, using the carbon monoxide (CO) and NO stretching bands as local probes of the active site of iNOS. Characteristic changes were observed in the spectra of the heme-bound ligands upon binding of the cofactors. Unlike photolyzed CO, which becomes trapped in well-defined orientations, as indicated by sharp photoproduct bands, photoproduct bands of NO photodissociated from the ferric heme iron were not visible, indicating that NO does not reside in the protein interior in a well-defined location or orientation. This may be favorable for NO release from the enzyme during catalysis because it reduces self-inhibition. Moreover, we used temperature derivative spectroscopy (TDS) with FTIR monitoring to explore the dynamics of NO and carbon monoxide (CO) inside iNOS after photodissociation at cryogenic temperatures. Only a single kinetic photoproduct state was revealed, but no secondary docking sites as in hemoglobins. Interestingly, we observed that intense illumination of six-coordinate ferrous iNOS oxy-NO ruptures the bond between the heme iron and the proximal thiolate to yield five-coordinate ferric iNOS oxy-NO, demonstrating the strong trans effect of the heme-bound NO.

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase

Inducible nitric oxide synthase (iNOS) is a homodimeric heme enzyme that catalyzes the formation of nitric oxide (NO) from dioxygen and L-arginine (L-Arg) in a two-step process. The produced NO can either diffuse out of the heme pocket into the surroundings or it can rebind to the heme iron and inhibit enzyme action. Here we have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy at cryogenic temperatures, using the carbon monoxide (CO) and NO stretching bands as local probes of the active site of iNOS. Characteristic changes were observed in the spectra of the heme-bound ligands upon binding of the cofactors. Unlike photolyzed CO, which becomes trapped in well-defined orientations, as indicated by sharp photoproduct bands, photoproduct bands of NO photodissociated from the ferric heme iron were not visible, indicating that NO does not reside in the protein interior in a well-defined location or orientation. This may be favorable for NO release from the enzyme during catalysis because it reduces self-inhibition. Moreover, we used temperature derivative spectroscopy (TDS) with FTIR monitoring to explore the dynamics of NO and carbon monoxide (CO) inside iNOS after photodissociation at cryogenic temperatures. Only a single kinetic photoproduct state was revealed, but no secondary docking sites as in hemoglobins. Interestingly, we observed that intense illumination of six-coordinate ferrous iNOSoxy-NO ruptures the bond between the heme iron and the proximal thiolate to yield five-coordinate ferric iNOSoxy-NO, demonstrating the strong trans effect of the heme-bound NO.

Measurement of energy barriers to conformational change in poly-l-glutamic acid by temperature-derivative spectroscopy

Energy barriers in kinetics are typically determined by measuring the rate as a function of temperature. We have used an alternative and complementary technique, temperature-derivative spectroscopy, to characterize folding of poly-l-glutamic acid in response to a change in pH. After the solution pH is changed photolytically at low temperature (150K) using a photogenerating acid, the solution is warmed while monitoring the helix–coil transition by its infrared absorption. At high initial pH, the secondary structure change occurs at 180K. If the initial pH is near 6, helix formation occurs near 190K. The results are interpreted in terms of the pre-exponential factor and enthalpy barrier for protein folding. Because the pH change is extrinsic to the system under study, this technique enables temperature-derivative spectroscopy to be used with any phenomenon that is pH dependent and with a wide range of spectroscopic measurement techniques.

Ligand Binding to Heme Proteins: A Comparison of Cytochrome c Variants with Globins

We have studied the binding of carbon monoxide (CO) in mutants of Cyt c having its methionine at position 80 replaced by alanine, aspartate, and arginine, so that the sixth coordination is available for ligand binding. We have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy to examine interactions of the heme-bound and photolyzed CO (and also nitric oxide, NO) in the small heme pocket created by the mutations. By using FTIR temperature derivative spectroscopy (TDS) and nanosecond flash photolysis, the enthalpy barrier distributions for CO rebinding were determined. In flash photolysis experiments, the majority of ligands rebind to the heme iron on picosecond time scales so that only the high-barrier tail of the distributions is visible on the nanosecond scale. By continuous wave excitation prior to TDS characterization of the barriers, however, each Cyt c molecule is photoexcited multiple times and complete photodissociation can be achieved, which likely arises from a rotation of the CO within the heme pocket so that the oxygen faces the heme iron. Apparently, reorientation prior to rebinding constitutes an additional and significant contribution to the rebinding barrier. Our experiments reveal that the compact, rigid structure of Cyt c offers no alternative binding sites for photodissociated ligands in the protein matrix. A comparison of ligand binding in these Cyt c mutants and hemoglobins underscores the importance of internal ligand docking sites and ligand migration routes for conveying a ligand binding function to heme proteins.

Spectroscopic Studies of Substrate and Ligand Binding in Inducible Nitric Oxide Synthase

Nitric oxide is a small molecule involved in various physiological processes. It serves for instance as a vasorelaxant or neurotransmitter. The three isoforms of nitric oxide synthase (eNOS, nNOS, iNOS) catalyze the formation of NO in a two-step reaction. In a first step arginine is converted to hydroxy-arginine. Subsequently, this intermediate is cleaved to yield citrulline and NO.In spite of the increasing interest in these proteins in medical research, little is known about the molecular details of the catalytic mechanism. We have used UV/visible and Fourier Transform Infrared / Temperature Derivative Spectroscopy (FTIR/TDS) over a wide range of temperature (4 - 300 K) to observe the interaction between protein (iNOS), ligand and substrate. In these experiments, the physiological ligand O2 has been replaced by CO to observe the ternary complex without steady turnover of arginine. In particular, we have focused on the influence of arginine, the intermediate hydroxy-arginine and cofactor H4B on ligand binding.

Temperature derivative spectroscopy to monitor the autoxidation decay of cytochromes P450.

Temperature derivative spectroscopy (TDS), a type of relaxation spectroscopy, is a powerful tool to study protein dynamics (Berendzen, J.; Braunstein, D. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 1). We developed the version of temperature derivative spectroscopy to monitor kinetics of autoxidation of cytochromes P450 and applied it to study the properties of the oxy-ferrous complex of a human membrane bound P450, CYP19A1 (aromatase), and that of a bacterial soluble P450, CYP101 when bound with their most common substrates, androstenedione (AD) and camphor, respectively. TDS extends the panel of methods that can be used to monitor heme protein kinetics, providing a rapid measurement technique and enabling measurement of the autoxidation rate over a wide range of temperatures, yielding the activation energy as well as absolute reaction rate in a single experiment.

Ligand Migration and Binding in Nonsymbiotic Hemoglobins of Arabidopsis thaliana

We have studied carbon monoxide (CO) migration and binding in the nonsymbiotic hemoglobins AHb1 and AHb2 of Arabidopsis thaliana using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures. Both proteins have similar amino acid sequences but display pronounced differences in ligand binding properties, at both physiological and cryogenic temperatures. Near neutral pH, the distal HisE7 side chain is close to the heme-bound ligand in the majority of AHb1-CO molecules, as indicated by a low CO stretching frequency at 1921 cm(-1). In this fraction, two CO docking sites can be populated, the primary site B and the secondary site C. When the pH is lowered, a high-frequency stretching band at approximately 1964 cm(-1) grows at the expense of the low-frequency band, indicating that HisE7 protonates and, concomitantly, moves away from the bound ligand. Geminate rebinding barriers are markedly different for the two conformations, and docking site C is not accessible in the low-pH conformation. Rebinding of NO ligands was observed only from site B of AHb1, regardless of conformation. In AHb2, the HisE7 side chain is removed from the bound ligand; rebinding barriers are low, and CO molecules can populate only primary docking site B. These results are interpreted in terms of differences in the active site structures and physiological functions.

Ligand Migration between Internal Docking Sites in Photodissociated Carbonmonoxy Neuroglobin

Neuroglobin (Ngb) belongs to the large family of globular heme proteins capable of binding small gaseous ligands such as O(2), CO, or NO within their active site. In this work, we have analyzed CO migration pathways in photolyzed NgbCO using molecular dynamics (MD) simulations in combination with Fourier transform infrared temperature derivative spectroscopy (FTIR-TDS). A total of 55 ns of MD simulation was analyzed to explore the approximately 300 A(3) internal Ngb cavity. Overall, the simulations differentiated between eight possible docking sites, three of which were also identified experimentally. Low-temperature FTIR-TDS experiments on wild-type (wt) and F28W mutant NgbCO revealed that a small fraction of ligands migrates from site B to site C from which they rebound after slow cool illumination. For the F28L mutant, however, population of site C was not observed. In agreement with these findings, the simulations at 20 K showed ligand transfer between sites B and C for wt Ngb, but not for the F28L mutant. The ligand migration network could be mapped out and two key gate residues, Phe28 and Pro52, were identified. Ligand population analysis from the MD simulations revealed a direct relation between the size of the B10 side chain (Phe28 in wild-type Ngb) and the barrier against migration. Barriers for the transition of photodissociated CO from the distal pocket to the Xe4 site in Ngb are lower by up to 4 kcal/mol compared to myoglobin, suggesting that ligand migration between different docking sites is more facile in Ngb than in myoglobin.

Influence of Proline on the Thermostability of the Active Site and Membrane Arrangement of Transmembrane Proteins

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro 50, Pro 91, and Pro 186 in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro 91, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro 91 and Pro 186 showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.

Structural Dynamics of Myoglobin: FTIR-TDS Study of NO Migration and Binding†

By using Fourier transform infrared photolysis difference spectroscopy combined with temperature derivative spectroscopy at cryogenic temperatures, we have measured infrared spectra of the stretching absorption on nitric oxide (NO) in the heme-bound and photodissociated states of ferrous and ferric nitrosyl myoglobin (MbNO) and a few site-specific Mb mutants. The NO absorption was utilized as a sensitive local probe of ligand interactions with active-site residues and movements within the protein. By comparison with results obtained in previous spectroscopic and structural studies of carbonmonoxy myoglobin (MbCO), the MbNO data were interpreted in structural terms. In the NO-bound state, conformational heterogeneity was inferred from the appearance of multiple bands, arising from different electrostatic interactions with active site residues, most importantly, His-64. In ferrous MbNO, a primary photoproduct site similar to site B of MbCO was found, as indicated by a characteristic NO stretching spectrum. In ferric MbNO, the His-64 side chain appears to interfere with trapping of NO in this site; only a very weak photoproduct spectrum was observed in Mb variants in which His-64 was present. Upon extended illumination, the photoproduct spectrum changed in a characteristic way, indicating that NO readily migrates to a secondary docking site C, the Xe4 cavity, in which the ligand performs librational motions on the picosecond time scale. This docking site may play a role in the physiological NO scavenging reaction. Surprisingly, NO cannot be trapped at all in secondary docking site D, the Xe1 cavity.

Influence of Distal Residue B10 on CO Dynamics in Myoglobin and Neuroglobin

For many years, myoglobin has served as a paradigm for structure-function studies in proteins. Ligand binding and migration within myoglobin has been studied in great detail by crystallography and spectroscopy, showing that gaseous ligands such as O(2), CO, and NO not only bind to the heme iron but may also reside transiently in three internal ligand docking sites, the primary docking site B and secondary sites C and D. These sites affect ligand association and dissociation in specific ways. Neuroglobin is another vertebrate heme protein that also binds small ligands. Ligand migration pathways in neuroglobin have not yet been elucidated. Here, we have used Fourier transform infrared temperature derivative spectroscopy at cryogenic temperatures to compare the influence of the side chain volume of amino acid residue B10 on ligand migration to and rebinding from docking sites in myoglobin and neuroglobin.

Ligand Migration and Binding in the Dimeric Hemoglobin ofScapharca inaequivalvis,

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of the dimeric hemoglobin of Scapharca inaequivalvis (HbI) after photodissociation. By combining these studies with X-ray crystallography, three transient ligand docking sites were identified: a primary docking site B in close vicinity to the heme iron, and two secondary docking sites C and D corresponding to the Xe4 and Xe2 cavities of myoglobin. To assess the relevance of these findings for physiological binding, we also performed flash photolysis experiments on HbICO at room temperature and equilibrium binding studies with dioxygen. Our results show that the Xe4 and Xe2 cavities serve as transient docking sites for unbound ligands in the protein, but not as way stations on the entry/exit pathway. For HbI, the so-called histidine gate mechanism proposed for other globins appears as a plausible entry/exit route as well.

The Position 68(E11) Side Chain in Myoglobin Regulates Ligand Capture, Bond Formation with Heme Iron, and Internal Movement into the Xenon Cavities

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.

Light-Induced Relaxation of Photolyzed Carbonmonoxy Myoglobin: A Temperature-Dependent X-Ray Absorption Near-Edge Structure (XANES) Study

X-ray absorption near-edge structure (XANES) spectra at the Fe K-edge have been measured and compared on solution samples of horse carbonmonoxy-myoglobin and its photoproducts, prepared by two different photolysis protocols: 1), extended illumination at low temperature (15 K) by white light; and 2), slow-cool from 140 to 10 K at a rate of 0.5 K/min while illuminating the sample with a 532-nm continuous-wave laser source. CO recombination has been followed while increasing the temperature at a rate of 1.2 K/min. After extended illumination at 15 K, a single process is observed, corresponding to CO recombination from a completely photolyzed species with CO bound to the primary docking site (formally B-state, in agreement with previous x-ray diffraction studies). The temperature peak for this single process is ∼50 K. Using slow-cool illumination, data show a two-state recombination curve, the two temperature peaks being roughly assigned to 50 K and 110 K. These results are in good agreement with previous FTIR studies using temperature-derivative spectroscopy. The XANES spectroscopic markers probe structural differences between the photoproduct induced by extended illumination at 15 K and the photoproduct induced by slow-cool illumination. These differences in the XANES data have been interpreted as due to light-induced Fe-heme relaxation that does not involve CO migration from the B-state. A quantitative description of the unrelaxed and relaxed B-states, including the measurements of the Fe-N p, Fe-N His, and Fe-CO distances, and the out-of-plane Fe displacement, has been obtained via a procedure (MXAN) recently developed by us. This work shows that XANES, being able to extract both kinetic and structural parameters in a single experiment, is a powerful tool for structural dynamic studies of proteins.

Structural Dynamics Controls Nitric Oxide Affinity in Nitrophorin 4

Nitrophorin 4 (NP4) is one of seven nitric oxide (NO) transporting proteins in the blood-sucking insect Rhodnius prolixus. In its physiological function, NO binds to a ferric iron centered in a highly ruffled heme plane. Carbon monoxide (CO) also binds after reduction of the heme iron. Here we have used Fourier transform infrared spectroscopy at cryogenic temperatures to study CO and NO binding and migration in NP4, complemented by x-ray cryo-crystallography on xenon-containing NP4 crystals to identify cavities that may serve as ligand docking sites. Multiple infrared stretching bands of the heme-bound ligands indicate different active site conformations with varying degrees of hydrophobicity. Narrow infrared stretching bands are observed for photodissociated CO and NO; temperature-derivative spectroscopy shows that these bands are associated with ligand docking sites close to the extremely reactive heme iron. No rebinding from distinct secondary sites was detected, although two xenon binding cavities were observed in the x-ray structure. Photolysis studies at approximately 200 K show efficient NO photoproduct formation in the more hydrophilic, open NP4 conformation. This result suggests that ligand escape is facilitated in this conformation, and blockage of the active site by water hinders immediate reassociation of NO to the ferric iron. In the closed, low-pH conformation, ligand escape from the active site of NP4 is prevented by an extremely reactive heme iron and the absence of secondary ligand docking sites.

Structural dynamics of myoglobin: an infrared kinetic study of ligand migration in mutants YQR and YQRF

Recombination of carbon monoxide to myoglobin mutants YQR and YQRF was studied using transient infrared absorption spectroscopy and Fourier transform infrared–temperature derivative spectroscopy (FTIR–TDS). Photoproduct states B, C′, C″ and D associated with ligands residing in different protein cavities have been identified. After photolysis, ligands migrate to primary docking site B and subsequently rebind or escape to a secondary site (C) within the Xe4 cavity. For YQR, a global analysis of the isothermal rebinding kinetics below 160 K and the TDS data reveal a correlation between the enthalpy barriers governing the two processes. Above 120 K, a protein conformational change in both YQR and YQRF converts photoproduct C′ into C″ with markedly slowed kinetics. Above ∼180 K, ligands migrate to the proximal Xe1 site (D) and also exit into the solvent, from where they rebind in a bimolecular reaction.

Structural Dynamics of Myoglobin: LIGAND MIGRATION AMONG PROTEIN CAVITIES STUDIED BY FOURIER TRANSFORM INFRARED/TEMPERATURE DERIVATIVE SPECTROSCOPY

Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A(0) and A(3), which can be distinguished by their different CO bands near 1965 and 1933 cm(-1). They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C', in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C") has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at approximately 180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C', C", and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C') is severely restricted by introduction of the bulky Phe side chain at position 107.

The Effect of Ligand Dynamics on Heme Electronic Transition Band III in Myoglobin

Band III is a near-infrared electronic transition at ∼13,000 cm −1 in heme proteins that has been studied extensively as a marker of protein conformational relaxation after photodissociation of the heme-bound ligand. To examine the influence of the heme pocket structure and ligand dynamics on band III, we have studied carbon monoxide recombination in a variety of myoglobin mutants after photolysis at 3 K using Fourier transform infrared temperature-derivative spectroscopy with monitoring in three spectral ranges, (1) band III, the mid-infrared region of (2) the heme-bound CO, and (3) the photodissociated CO. Here we present data on mutant myoglobins V68F and L29W, which both exhibit pronounced ligand movements at low temperature. From spectral and kinetic analyses in the mid-infrared, a small number of photoproduct populations can be distinguished, differing in their distal heme pocket conformations and/or CO locations. We have decomposed band III into its individual photoproduct contributions. Each photoproduct state exhibits a different “kinetic hole-burning” (KHB) effect, a coupling of the activation enthalpy for rebinding to the position of band III. The analysis reveals that the heme pocket structure and the photodissociated CO markedly affect the band III transition. A strong kinetic hole-burning effect results only when the CO ligand resides in the docking site on top of the heme group. Migration of CO away from the heme group leads to an overall blue shift of band III. Consequently, band III can be used as a sensitive tool to study ligand dynamics after photodissociation in heme proteins.