Taegon Lee

Dynamics of ultrafast rebinding of CO to carboxymethyl cytochrome c.

Rebinding dynamics of CO to carboxymethyl cytochrome c (Ccytc), a chemically modified cytochrome c to bind ligands in its ferrous form, in D(2)O solution at 283 K after photodeligation, was investigated using femtosecond vibrational spectroscopy. The stretching mode of CO bound to the protein shows four stretching bands near 1962 cm(-1). Time-resolved spectra of the bound CO revealed a slight band-position-dependent rebinding kinetics, suggesting that the geminate rebinding of CO depends on the conformation of the protein. The overall rebinding kinetics of CO to Ccytc was more than 1000 times faster than that to myoglobin (Mb), a ligand-binding protein, and is also faster than a model heme, microperoxidase-8 in viscous solvent. The efficient rebinding of CO to Ccytc was attributed to the longer retention of the dissociated CO near the active binding site by the organized protein matrix of Ccytc. The spectra of the dissociated CO reveal a fast-growing band in the picosecond time scale that is assigned to CO in D(2)O solvent. The ultrafast CO escape to bulk solution is consistent with its 3D structure showing a sizable opening in the active site. It appears that most of the dissociated CO rebinds within 1 ns, except for those that escape to the bulk solution through the opening. The CO rebinding in Ccytc indicates that the primary heme pocket in Mb, located near the active site and holding the dissociated ligand for longer than tens of nanoseconds, has a specific structure to suppress CO rebinding.

Dynamics of Geminate Rebinding of NO with Cytochrome c in Aqueous Solution Using Femtosecond Vibrational Spectroscopy

Using femtosecond vibrational spectroscopy, we investigated the rebinding dynamics of NO to cytochrome c (Cytc) and a model heme, microperoxidase-8 (Mp), after photodeligation of CytcNO in D(2)O solution and MpNO in an 81% glycerol/water (v/v) mixture at room temperature. Whereas the stretching mode of the NO band in MpNO was described by a Gaussian centered at 1653 cm(-1) with a full width at half-maximum (fwhm) of 41 cm(-1), that in CytcNO revealed an asymmetric structured band that peaked at 1619 cm(-1) with an fwhm of about 27 cm(-1). The structured NO band in CytcNO was well described by the sum of three Gaussians, and its shape did not evolve with time but its amplitude decayed exponentially with a time constant of 7 ± 1 ps. The transient NO band in MpNO also decayed exponentially with a time constant of 8 ± 1 ps. Rebinding of NO to Cytc was slightly faster than that of NO to Mp and was almost complete by 30 ps, which was much faster than the rebinding of NO to myoglobin (Mb). When the deligated NO was constrained near the Fe atom either by a viscous solvent or by the protein matrix, it rebound to heme Fe much faster than CO, suggesting that NO has a higher propensity for binding to heme Fe and the high reactivity governed the rebinding kinetics. Moreover, the faster ligand rebinding in Cytc than in Mb suggests that Cytc does not have a primary docking site (PDS)-like structure found in Mb that suppresses rebinding by restraining ligand motion and the PDS can also hold the deligated NO in a manner that impedes NO rebinding; however, due to higher NO reactivity with heme Fe, the impediment is not as efficient as for CO.

Direct observation of ligand rebinding pathways in hemoglobin using femtosecond mid-IR spectroscopy.

The dynamics of NO rebinding in hemoglobin (Hb) was directly observed using femtosecond mid-IR spectroscopy after photodeligation of NO from HbNO in D(2)O at 283 K. Time-resolved spectra of bound NO appeared to have a single feature peaked at 1616 cm(-1) but were much better described by two Gaussians with equal intensities but different rebinding kinetics, where the feature at 1617 cm(-1) rebinds faster than the one at 1614 cm(-1). It is possible that the two bands each correspond to one of two subunit constituents of the tetrameric Hb. Transient absorption spectra of photodeligated NO revealed three evolving bands near 1858 cm(-1) and their red-shifted replicas. The red-shifted replicas arise from photodeligated NO in the vibrationally excited v = 1 state. More than 10% of the NO was dissociated into the vibrationally excited v = 1 state when photolyzed by a 580 nm pulse. The three absorption bands for the deligated NO could be attributed to three NO sites in or near the heme pocket. The kinetics of the three transient bands for the deligated NO, as well as the recovery of the bound NO population, was most consistent with a kinetics scheme that incorporates time-dependent rebinding from one site that rapidly equilibrates with the other two sites. The time dependence results from a time-dependent rebinding barrier due to conformational relaxation of protein after deligation. By assigning each absorption band to a site in the heme pocket of Hb, a pathway for rebinding of NO to Hb was proposed.

A Description of Vibrational Modes in Hexaphyrins: Understanding the Aromaticity Reversal in the Lowest Triplet State

Aromaticity reversal in the lowest triplet state, or Bairds rule, has been postulated for the past few decades. Despite numerous theoretical works on aromaticity reversal, experimental study is still at a rudimentary stage. Herein, we investigate the aromaticity reversal in the lowest excited triplet state using a comparable set of [26]- and [28]hexaphyrins by femtosecond time-resolved infrared (IR) spectroscopy. Compared to the relatively simple IR spectra of [26]bis(rhodium) hexaphyrin (R26H), those of [28]bis(rhodium) hexaphyrin (R28H) show complex IR spectra the region for the stretching modes of conjugated rings. Whereas time-resolved IR spectra of R26H in the excited triplet state are dominated by excited state IR absorption peaks, while those of R28H largely show ground state IR bleaching peaks, reflecting the aromaticity reversal in the lowest triplet state. These contrasting IR spectral features serve as new experimental aromaticity indices for Bairds rule.

Photoexcitation Dynamics of NO-Bound Ferric Myoglobin Investigated by Femtosecond Vibrational Spectroscopy

Femtosecond vibrational spectroscopy was used to investigate the photoexcitation dynamics of NO-bound ferric myoglobin (Mb(III)NO) in D2O solution at 294 K after excitation with a 575 nm pulse. The stretching mode of NO in Mb(III)NO consists of a major band at 1922 cm(-1) (97.7%) and a minor band at 1902 cm(-1) (2.3%), suggesting that Mb(III)NO in room temperature solution has two conformational substates. The time-resolved spectra show small but significant new absorption features at the lower-energy side of the main band (1920-1800 cm(-1)). One new absorption feature in the region of 1920-1880 cm(-1) exhibits the (15)NO isotope shift (37 cm(-1)) the same as that of the NO band in the ground electronic state of Mb(III)NO. This absorption shifts toward higher energy and narrows with a time constant of 2.4 ps, indicating that it evolves with rapid electronic and thermal relaxation of the photoexcited Mb(III)NO without photodeligation of the NO from the heme. Absorption features assigned to proteins undergoing thermal relaxation without NO deligation add up to 14 ± 1% of the total bleach, implying that the photolysis quantum yield of Mb(III)NO with a Q-band excitation is ≤0.86 ± 0.01. The remaining absorption bands peaked near 1867, 1845, and 1815 cm(-1), each showing the (15)NO isotope shift the same as that of the free NO radical (33 cm(-1)), were assigned to the vibrational band of the photodeligated NO, the NO band of Mb(III)NO in an intermediate electronic state with low-spin Fe(III)-NO(radical) character (denoted as the R state), and the NO band of the vibrationally excited NO in the R state, respectively. A kinetics model successfully reproducing the time-dependent intensity changes of the transient bands suggests that every rebound NO forms the R state that eventually relaxes into the ground electronic state nonexponentially. Most of the photodissociated NO undergoes fast geminate recombination (GR), and the rebinding kinetics depends on the conformation of the protein. GR of NO to Mb(III) in the major conformation shows highly nonexponential kinetics described by a stretched exponential function, exp(-(t/290 ps)(0.44). The NO rebinding to Mb(III) in the minor conformation is exponential, exp(-t/1.8 ns), suggesting that the distal histidine, the interaction of which dictates the conformation of Mb(III)NO, participates in mediating the binding of NO to Mb(III). In Mb(III)NO, the elusive low-spin Fe(III)-NO(radical) state, proposed in electronic structure calculations, indeed exists at >12 kJ/mol above the ground state and takes part in the bond formation of Fe(III)-NO, suggesting that it plays a significant role in the function of NO-bound ferric protein. Time-resolved vibrational spectra with high sensitivity reveal rich photophysical and photochemical processes of photoexcited Mb(III)NO.

Viscosity-dependent dynamics of CO rebinding to microperoxidase-8 in glycerol/water solution.

Rebinding kinetics of CO to microperoxidase-8 (Mp), an excellent model system for the active site of heme proteins such as myoglobin and hemoglobin, was measured after photolysis of MpCO in solutions with various viscosities and temperatures, using femtosecond vibrational spectroscopy. Whereas the geminate rebinding of CO to Mp in water is negligible, significant fractions of CO rebind nonexponentially within 1 ns at room temperature in a glycerol/water solution. The geminate yield of the CO rebinding increases and its rate accelerates as the viscosity of the solution increases either by increasing glycerol content in glycerol/water mixtures at 294 K or by decreasing temperature of the solution from 323 to 283 K. The nonexponential rebinding kinetics can be described by the theory of a diffusion-controlled reaction and the data are well reproduced by the pair survival probability function in the absence of any interaction potential between the pair. The rebinding kinetics was also successfully described by the SRC model, a distributed linear coupling model for the CO rebinding.

Dynamics of ligand rebinding to unfolded MbCO by guanidine HCl.

Femtosecond vibrational spectroscopy was used to probe a functionally important dynamics and residual structure of myoglobin unfolded by 4 M guanidine HCl. The spectra of the dissociated CO indicated that the residual structure of unfolded myoglobin (Mb) forms a few hydrophobic cavities that could accommodate the dissociated ligand. Geminate rebinding (GR) of CO to the unfolded Mb is three-orders-of-magnitude faster and more efficient than the native Mb but similar to a model heme in a viscous solvent, suggesting that the GR of CO to heme is accelerated by the longer retention of the dissociated ligand near the Fe atom by the poorly-structured protein matrix of the unfolded Mb or viscous solvent. The inefficient GR of CO in native Mb, while dissociated CO is trapped in the primary heme pocket located near the active binding site, indicates that the tertiary structure of the pocket in native Mb plays a functionally significant role.

Direct Observation of the Low-Spin Fe(III)–NO(radical) Intermediate State during Rebinding of NO to Photodeligated Ferric Cytochrome c

Nitrosylated ferric heme is autoreduced readily to the more stable Fe(II)-NO adduct, but it is stabilized in NO-carrier heme proteins where maintaining the Fe(III) oxidation state is crucial for efficient NO delivery. Density functional theory calculations by Lehnert and co-workers have shown that a NO-bound ferric model heme has a low-spin (LS) Fe(III)-NO(radical) state that might be critical for efficient NO transport by NO-carrier heme proteins. Recently, the elusive LS Fe(III)-NO(radical) state was observed as an electronic intermediate state during geminate rebinding (GR) of NO to ferric myoglobin (Mb(III)). Cytochrome c (Cytc), a ubiquitous heme protein, is useful for generalizing the presence of the LS Fe(III)-NO(radical) state. Photoexcitation dynamics of NO-bound ferric Cytc (Cytc(III)NO) was probed after excitation of Cytc(III)NO in D2O solution at 294 K with a 575 nm pulse using femtosecond vibrational spectroscopy. The time-resolved spectra displayed several weak absorption bands in the 1900-1800 cm(-1) range and a dominant bleach at 1917 cm(-1), the position of the absorption at equilibrium. Two absorptions, with 37 cm(-1) isotope shift of (15)NO, shifted toward higher energy and narrowed with an average time constant of 8 ps, indicating that they arose from thermally and/or vibrationally excited NO in the ground electronic state of Cytc(III)NO. Three absorption bands, showing 33 cm(-1) isotope shift of (15)NO and peaked at 1865, 1836, and 1807 cm(-1), were assigned to the deligated NO residing in the interior of the protein, to the rebound Cytc(III)NO in the LS Fe(III)-NO(radical) state, and to the vibrationally excited NO of Cytc(III)NO in the LS Fe(III)-NO(radical) state, respectively. The quantum yield for NO deligation of Cytc(III)NO by a 575 nm photon was 0.8 ± 0.1. Most of the deligated NO showed non-exponential GR, and the GR kinetics was described by exp(-(t/7 ps)(0.7)). Every rebound Cytc(III)NO formed the LS Fe(III)-NO(radical) state that relaxed into the ground state, with the relaxation kinetics described by exp(-(t/2.5 ps)(0.7)). The GR of NO to ferric Cytc was as fast as the thermal relaxation of hot heme, and the relaxation of the rebound Cytc(III)NO in the intermediate LS Fe(III)-NO(radical) state was faster than the thermal relaxation of hot heme, generating the rebound Cytc(III)NO in a thermally excited ground electronic state. For both Cytc(III)NO and Mb(III)NO, the relaxation rate of the LS Fe(III)-NO(radical) state was similar to the upper rate limit of the domed-to-planar heme transition observed in NO-rebound ferrous-heme proteins, suggesting that the change in the Fe-NO bond length is coupled to the doming motion of the heme Fe.

Probing the Role of Hydration in the Unfolding Transitions of Carbonmonoxy Myoglobin and Apomyoglobin

We show that the equilibrium unfolding transition of horse carbonmonoxy myoglobin monitored by the stretching vibration of the CO ligand, a local environmental probe, is very sharp and, thus, quite different from those measured by global conformational reporters. In addition, the denatured protein exhibits an A(0)-like CO band. We hypothesize that this sharp transition reports penetration of water into the heme pocket of the protein. Parallel experiments on horse apomyoglobin, wherein an environment-sensitive fluorescent probe, nile red, was used, also reveals a similar putative hydration event. Given the importance of dehydration in protein folding and also the recent debate over the interpretation of probe-dependent unfolding transitions, these results have strong implications on the mechanism of protein folding.

Dynamics of Geminate Rebinding of CO to Cytochrome c in Guanidine HCl Probed by Femtosecond Vibrational Spectroscopy

Femtosecond vibrational spectroscopy was used to probe the rebinding dynamics of CO to cytochrome c (Cytc) in 1.8 and 7 M guanidine HCl (GdnHCl) after photodeligation of the corresponding CO-bound protein in D2O buffer (pD = 7.4) at 283 K. Geminate rebinding (GR) dynamics of CO to the folded Cytc in 1.8 M GdnHCl (nCytc) is similar to that to chemically modified cytochrome c (cCytc), suggesting that the overall conformations of nCytcCO and cCytcCO are similar. About 86% of the dissociated CO molecules were geminately rebound to nCytc nonexponentially within 1 ns. The efficient GR of CO to the folded Cytc can be attributed to the organized protein matrix near the active site of nCytc that provides an efficient trap for the diffusing CO ligand after photodissociation. Although the concentration of nCytc did not affect its GR yield of CO, GR yield of CO to the unfolded Cytc in 7 M GdnHCl (uCytc) increased from 5 to 30% as the protein concentration increased from 0.3 to 9 mM. Time-resolved spectra of the (13)CO dissociated from both 9 mM nCytc(13)CO and 9 mM uCytc(13)CO showed a growing band with a peak at 2090 cm(-1) on the picosecond time scale, which was assigned to (13)CO in D2O solvent. At 1 ns, the fraction of the CO band in the solvent was about 10% of the nascent photodeligated protein in nCytc and more than 50% in the concentrated uCytc. Whereas a small opening in the active site of nCytc is responsible for the ultrafast escape of CO to solution in the folded protein, a large fraction of the CO escape to the solvent in uCytc results from the denatured structure of the active site in the unfolded protein. The spectrum of the CO dissociated from the concentrated uCytcCO contained a band that decayed as efficiently as that for the folded protein, suggesting that some fraction of uCytcCO might form aggregates even in 7 M denaturant, such that the aggregate acts as an efficient trap for the diffusing CO after deligation. No hint of precipitate in the concentrated uCytcCO and protein refolding upon dilution of the GdnHCl indicate that the aggregate does not grow continuously but remains as a soluble oligomer. The delayed appearance of the solvated CO and the inefficient GR of CO in uCytcCO suggest that the monomeric unfolded CytcCO so loosely arranged that the protein matrix cannot trap CO efficiently but the bound CO is still buried within hydrophobic residues even under the harsh denaturation condition.