Biomedical subjects

P M Champion

Publications and source records attributed to P M Champion.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119.

Using UV-Vis, resonance Raman, and EPR spectroscopy we have studied the properties of the oxygenated ferrous cytochrome P450 from Sulfolobus solfataricus, (CYP119). The recently determined crystal structure of CYP119 is compared with other available structures of P450s, and detailed structural and spectroscopic analyses are reported. With several structural similarities to CYP102, such as in-plane iron position and a shorter iron-proximal ligand bond, CYP119 shows low-spin conformation preference in the ferric form and partially in the ferrous form at low temperatures. These structural features can explain the fast autoxidation of the oxyferrous complex of CYP119. Finally, we report the first UV-Vis and EPR spectra of the cryoradiolytically reduced oxygenated intermediate of CYP119. The primary reduced intermediate, a hydroperoxo-ferric complex of CYP119, undergoes a 'peroxide shunt' pathway during gradual annealing at 170-195 K and returns to the low-spin ferric form.

Archaeal Proteins↗

PubMed2001

Resonance Raman and EPR investigations of the D251N oxycytochrome P450cam/putidaredoxin complex.

We have performed resonance Raman and electron paramagnetic resonance (EPR) studies on the dioxygen bound state of the D251N mutant of cytochrome P450cam (oxy-P450cam) and its complex with reduced putidaredoxin (Pd). The D251N oxy-P450cam/Pd complex has a perturbed proton delivery mechanism and shows a significantly red-shifted UV-visible spectrum as observed in Benson et al. [Benson, D. E., Suslick, K. S., and Sligar, S. G. (1997) Biochemistry 36, 5104-5107]. The red shift has been interpreted to indicate a major perturbation of the electronic structure of the oxy-heme complex. However, we find no evidence that electron transfer has occurred from Pd to the heme active site of D251N oxy-P450cam. This suggests that both electron and proton transfer are perturbed by the D251N mutation and that these processes may be coupled. Three oxygen isotope sensitive Raman features are identified in the Pd complex, and occur at 1137, 536, and 399 cm(-1). These values are not significantly different from those for WT or D251N oxy-P450cam. However, a careful examination of the oxygen stretching feature near 1137 cm(-1) reveals the presence of three peaks at 1131, 1138, and 1146 cm(-1), which we attribute to the presence of conformational substates in oxy-P450cam. A significant change in the conformational substate population is observed for the D251N oxy-P450cam when the Pd complex is formed. We suggest that the conformational population redistribution of oxy-P450cam, along with the red-shifted electronic spectra, reflects a structural equilibrium of the oxy-heme that is perturbed upon Pd binding. We propose that this structural perturbation is connected to the effector function of Pd and may involve changes in the electron donation properties of the thiolate ligand.

Amino Acid Substitution↗

PubMed2001

Long-range reactive dynamics in myoglobin.

We report the complete vibrational spectrum of the probe nucleus 57Fe at the oxygen-binding site of the protein myoglobin. The Fe-pyrrole nitrogen stretching modes of the heme group, identified here, probe asymmetric interactions with the protein environment. Collective oscillations of the polypeptide, rather than localized heme vibrations, dominate the low frequency region. We conclude that the heme "doming" mode is significantly delocalized, so that distant sites respond to oxygen binding on vibrational time scales. This has ramifications for understanding long-range interactions in biomolecules, such as those that mediate cooperativity in allosteric proteins.

Animals↗

PubMed2001

Water penetration and binding to ferric myoglobin.

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.

Amino Acid Substitution↗

PubMed1997

Spectroscopic effects of polarity and hydration in the distal heme pocket of deoxymyoglobin.

Distal pocket mutations at the E7 position (His64) of sperm whale deoxymyoglobin (deoxyMb) are used as a probe of distal pocket polarity and hydration. Changes of two key spectroscopic markers, the Fe-His(F8) stretch in the resonance Raman spectrum and the position of band III in the absorption spectrum, are monitored as the His64Tyr, His64Phe, His64Leu, and His64Gly mutations alter the distal heme pocket environment. The Fe-His vibration for the Phe, Leu, and Gly mutants is shifted to a lower frequency by 1-2 cm-1 relative to the Tyr mutant, wild type (WT), and native deoxyMb. Band III shifts to the red by approximately 4 nm ( approximately 70 cm-1) relative to WT and native deoxyMb for all the His64 mutants examined in this work. We correlate the small shift in the Fe-His frequency to the local electrostatic environment directly above the heme iron, affected by the presence of a localized water molecule in the heme pocket that is hydrogen-bonded to the E7 residue. The position of band III is roughly correlated to the displacement of the iron from the heme plane; however, the relatively large scatter in this correlation, as well as its dependence on distal pocket mutations, suggests that the heme pocket environment, particularly the E7 residue, also affects the energy of this transition.

Animals↗

PubMed1996

Probing heme protein conformational equilibration rates with kinetic selection.

Double-pulse flash photolysis experiments on solutions of carbonmonoxymyoglobin (MbCO) are used to determine the time scale for protein conformational averaging. The interconversion times for transitions between the "open" and "closed" subpopulations of MbCO are found to be 10(-6)-10(-4)s, depending on solvent composition and temperature. In aqueous solution at 273 K, the interconversion rate is found to be 1.4 x 10(6)s. Since the interconversion rate is comparable to or slower than the geminate rebinding rate, we describe the geminate phase of the kinetics as a superposition of contributions from the open and closed states. Although geminate kinetics remain intrinsically nonexponential for both open and closed states near room temperature, we find that substates within these two subpopulations interconvert more rapidly than the geminate rebinding. These observations cannot be explained by a superposition of contributions from a quasicontinuous conformational distribution (Steinbach et al., 1991) and are probably due to the long-time tail of the relaxation of the protein (Tian et al., 1992). Bimolecular rebinding takes place at a statistically averaged rate, since the interconversion and relaxation rates are faster than the bimolecular kinetics. The geminate and bimolecular kinetics are analyzed quantitatively as a function of pH using this approach and the spectroscopically determined populations of the open and closed states. The analysis accounts for the observed kinetics and also successfully predicts the kinetic response observed in the double-pulse experiments. In aqueous solution at 273 K, the geminate amplitudes and rates are found to be I(0)g = 32% and k(0)g = 1.3 x 10(7)s(-1) for the open state and I(1)g = 9.3% and k(1)g = 1.4 x 10(6)s(-1) for the closed state. In 75% glycerol solution at 264 K, the dominant component of the geminate rebinding is characterized by I(0)g1 = 89% and k(0)g1 = 3.1 x 10(6)s(-1) for the open state and I(1)g1 = 26% and k(1)g1 = 3.1 x 10(6)s(-1) for the closed state. The fact that the interconversion rate is comparable to the geminate rate of the closed state in aqueous solution is consistent with the idea that the open state provides an important pathway for ligand escape from (or entry to) the heme pocket (Tian et al., 1993). The increased viscosity of 75% glycerol solution delays the closed--> open interconversion until the end of the geminate phase, which forces the ligand to find alternative pathways to the solution. This observation, in conjunction with the near equivalence of the geminate rates for the open and closed states in 75% glycerol solution, suggests that the solvent composition fundamentally alters the protein-ligand dynamics.

Animals↗

PubMed1995

Measurements of CO geminate recombination in cytochromes P450 and P420.

The kinetics of CO geminate recombination in cytochrome P450cam are studied at room temperature subsequent to laser photolysis. The geminate rebinding kinetics of P450 are strongly affected by the presence of the camphor substrate. We observe a approximately 2% geminate yield for substrate-bound P450 and a 90% geminate yield when the substrate is absent. The drastic difference in the geminate kinetics suggests that the presence of camphor significantly alters the CO rebinding and escape rates by modifying the heme pocket environment. Two geminate phases and two bimolecular rebinding phases in the substrate free protein were observed, which could arise from slowly interconverting protein conformations. When the temperature or the viscosity of the solution is changed, the fast geminate rate remains the same, whereas the slow geminate rate and the two bimolecular rates change significantly. The geminate rebinding yield of substrate-free P420 is smaller than that of substrate free P450, but its geminate rebinding rate is faster. This demonstrates that in the absence of substrate, CO escapes from the pocket of P420 much more rapidly than from P450 and suggests that the distal pocket environment is altered in the P420 form.

Carbon Monoxide↗

PubMed1994

Observation of coherent reaction dynamics in heme proteins.

Femtosecond laser pulses, resonant with Soret band of the nitric oxide complex of myoglobin (MbNO), were used to probe coherent, low-frequency nuclear motion of the heme group after photolysis. Distinct oscillations with periods of 430 and 150 femtoseconds were observed and are attributed to heme doming and iron-histidine motion, respectively. These results verify that the nuclear motion of the heme is strongly coupled to the ligand binding reaction and demonstrate that such motion is not determined by overdamped (diffusive) dynamics. The relative phases and frequencies of the nuclear motion of the photoproduct suggest that the coherence arises from impulsive electronic forces associated with the spin-state change of the heme iron atom and the depopulation of its dz2 orbital during the bond-breaking event.

Histidine↗

PubMed1994

Investigations of the thermal response of laser-excited biomolecules.

A model is presented that connects the underlying classical thermal transport coefficients to the experimentally determined vibrational temperature of a photoexcited chromophore embedded in a protein matrix that is surrounded by water. Both photo-stationary state heating (e.g., within a 10-ns laser pulse) and transient cooling (e.g., after termination of the laser pulse) are treated. Because only a few thermal transport parameters can be experimentally determined, this simple model provides a practical and efficient method for describing the temperatures of the chromophore, protein, and solvent as functions of time and position. We expect that such a model will be useful in interfacing experimental observations with more elaborate molecular dynamics calculations, which depend upon many variables. In the transient cooling process, which is relevant for ultrafast pulsed laser measurements, the temperature of the chromophore follows a double exponential decay at short times, whereas at longer times the thermal decay "rolls over" to a diffusion limit (t-3/2). For typical 10-ns laser pulses (approximately 0.5 GW/cm2) and chromophore absorption cross-sections (approximately 10(-16) cm2), we find that the biomolecule reaches thermal steady-state on a ps time scale. The role of the various thermal transport coefficients and their independent experimental determination is also discussed.

Biophysical Phenomena↗

PubMed1993

Quantitative structural comparisons of heme protein crystals and solutions using resonance Raman spectroscopy.

Resonance Raman difference spectra have been used to compare crystal and solution samples of metmyoglobin (metMb), deoxymyoglobin (deoxyMb), and cytochrome P450. At pH 6.0, the frequency shifts of the heme core size sensitive bands v2, v3, and v4 were determined to be less than 0.3, 1.0, and 0.3 cm-1, respectively, for metMb and to be less than 1.0, 1.0, and 0.3 cm-1, respectively, for deoxyMb. This shows that the heme core size differences between the crystal and solution conformations are less than 0.002 A for metMb and less than 0.003 A for deoxyMb. These results disagree with a recent extended X-ray absorption fine structure study [Zhang, K., Chance, B., Reddy, K. S., Ayene, I., Stern, E. A., & Bunker, G. (1991) Biochemistry 30, 9116-9120] which claims that a 0.05-A difference exists in the average iron-ligand distance between the crystalline and solution forms of metMb at pH 6.5. At pH 8.5, metMb solution samples change gradually from a predominantly high-spin to a predominantly low-spin species as the ammonium sulfate concentration is increased to the level found in the crystal mother liquor. No Raman frequency shifts are found between the crystal and solution forms of metMb at pH 8.5 when the ammonium sulfate concentrations are equal. On the other hand, for deoxyMb, we find a significant alteration in the 220/240-cm-1 line shape and relative intensities, suggesting that some histidine-heme perturbation takes place upon crystallization.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Sulfate↗

PubMed1993

Investigations of ligand association and dissociation rates in the "open" and "closed" states of myoglobin.

Kinetic and Raman spectroscopic studies are combined to analyze ligand association and dissociation rates as a function of pH in aqueous solutions of myoglobin. A double-pulse flash photolysis protocol is used to kinetically select a rapidly rebinding (open pocket) fraction of the myoglobin ensemble and determine the timescale for averaging (approximately 1 to 10 microseconds) between the "open" and "closed" distal pocket protein conformations. Since this timescale is fast compared to the rate of ligand migration from the solution to the heme pocket (approximately 10(-4)s), a time-averaged population analysis, rather than a superposition of states, can be used to describe the ligand association and dissociation kinetics. Raman spectroscopy provides the relative populations of the open and closed distal pocket states as a function of pH which, in parallel with kinetics measurements, are used to determine the rates for ligand association and dissociation specific to these states. In aqueous solution at 293 K (1 mM CO) we find kon0 = 5.6 x 10(3) s-1, koff0 = 8.5 x 10(-2) s-1 for the open state and kon1 = 5.0 x 10(2) s-1, koff1 = 1.3 x 10(-2) s-1 for the closed state. The order of magnitude increase in the dissociation and association rates of the open form suggests that it may play a significant role in the ligand binding process, even though it comprises only approximately 5% of the time-averaged population at pH 7. For oxygen binding at 293 K (1.36 mM O2) we find kon0 = 4.6 x 10(4) s-1, koff0 approximately 10(4 +/- 2) s-1 for the open state and kon1 = 2.0 x 10(4) s-1, koff1 = 13 s-1 for the closed state. The dramatic increase in the dissociation rate of the open form is probably due to the loss of the hydrogen bond with the distal histidine, which stabilizes the bound O2 in the closed state. Overall, these results demonstrate that the open conformation plays a significant role in determining the ligand association and dissociation rates and suggest that environmentally induced modulations of the open population could be used as a biomolecular control mechanism for the uptake and delivery of oxygen in muscle cells.

Carbon Monoxide↗

PubMed1992

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.

Bacterial Proteins↗

PubMed1992

Conformational interconversion in protein crystals.

We present evidence that the structure of carbonmonoxy myoglobin crystals can be altered by lowering the pH. This structural change is monitored by the characteristic Fe-CO Raman modes at 508 and 491 cm-1 and is thought to involve a localized distal pocket transition from a "closed" conformation at pH 7 to a more "open" conformation at pH 4. These changes take place in the crystal without loss of intensity of a conformationally sensitive Raman mode at 252 cm-1 that signals a partial unfolding of the globin structure in solution. Quantitative studies, which monitor the open and closed populations as a function of laser photolysis, demonstrate that the interconversion rates (k+/-) in solution at 298 K are fast compared to the photolysis and CO entry rates (i.e. k+/- much greater than 10(3) s-1), while in frozen samples the interconversion is much slower than the experimental time scale (minutes). Since the open conformation is a minority species at pH 7, rapid exchange in aqueous solution is a necessary condition for this species to play a functional role. In the crystal, the interconversion rates are slowed compared to solution and begin to approach the photolysis rate (i.e. k+/- approximately 10(3) to 10(4) s-1). This indicates that the barriers for conformational exchange are increased in the crystal environment, compared to the solution, apparently due to the packing forces of the surrounding molecules. X-ray and neutron diffraction studies of MbCO crystals at high and low pH are needed to characterize the details of the structural changes and to test the hypothesis that closed and open distal pocket structures are associated with the 508 and 491 cm-1 Fe-CO modes.

Hydrogen-Ion Concentration↗

PubMed1992

Low pH myoglobin photoproducts.

Recently, there has been interest in determining the conditions under which the iron-histidine bond ruptures in myoglobin at low pH, so that the effect of proximal heme ligation can be studied. A 220-cm-1 Raman mode, assigned to iron-histidine stretching, is clearly visible after photolysis of aqueous MbCO samples below pH4 at room temperature (Sage et al. Biochemistry. 30:1237-1247). In contrast, Iben et al. (Biophys. J. 59:908-919) do not observe this mode upon photolysis of a pH3 MbCO sample in a glycerol/water glass at low temperature. In order to account for both the low temperature and the room temperature experiments, Iben et al. suggest a scheme involving an unusual protonation state of the proximal histidine. Here, we discuss some inconsistencies in their explanation of the room temperature results and offer instead a simple modification of an earlier model. In addition, circular dichroism data are presented that indicate partial unfolding of MbCO in aqueous solution below pH4, and raise questions about the claim of Iben et al. that MbCO remains folded in 75% glycerol at pH3.

Biophysical Phenomena↗

PubMed1991

Investigations of optical line shapes and kinetic hole burning in myoglobin.

We present the results of an extensive investigation of the optical line shapes of deoxymyoglobin (Mb), the ligand-bound form (MbCO), and the low-temperature photoproduct (Mb*). The thermal properties and the pH dependence of the Soret band and the near infrared band III (approximately 760 nm) are analyzed, taking into account the underlying vibrational properties of the absorption bands. The strong temperature dependence associated with the Soret band of MbCO and band III of Mb indicates significant coupling to low-frequency modes that may not be directly observed in the resonance Raman spectra. On the basis of analogous line-shape studies in a variety of heme systems, we assign the low-frequency coupling in MbCO to torsional motions of the CO molecule. The low-frequency mode coupled to band III (approximately 70 cm-1) is found to lie quite close to the value for the heme-doming motion (approximately 50 cm-1) calculated by using the kinetically determined value of the force constant (17 N/m). Significant inhomogeneous broadening in the Soret region of Mb and Mb* is found to be due to a "nonkinetic" coordinate that we associate with the orientation of the proximal histidine. A "kinetic" coordinate, associated with the equilibrium displacement of the iron atom from the porphyrin plane (a) is found to contribute to the inhomogeneous broadening of both the Soret band and band III. The relaxation of the heme as the system evolves from from Mb* to Mb is followed optically as a function of temperature, and a sharp transition temperature is found at 185 K. The blue shifts of the Soret band and band III as Mb* evolves to Mb are found to be nearly identical (delta v*ABS approximately 140 cm-1) and attributed to changes in the mean value of a between Mb* (a*0) and Mb (a0 = 0.45 A). A simple quadratic model for the coordinate coupling that simultaneously accounts for the observed shift, delta v*ABS, the low-temperature kinetics and the kinetic hole burning predicts a*0 = 0.2 +/- 0.05 A and EA = 16 +/- 2 kJ/mol for the room temperature Arrhenius barrier height at the heme. A simple quantitative method for the analysis of kinetic hole-burning experiments is also developed and applied to recent studies involving quaternary and subunit-specific hemoglobin structures.

Carbon Monoxide↗

PubMed1991

Investigation of laser-induced long-lived states of photolyzed MbCO.

We present evidence from resonance Raman and absorption measurements that the extended exposure of MbCO to CW laser light at low temperatures alters the CO rebinding kinetics and leads to a significantly increased population of very long lived states of photolyzed MbCO. This optical "pumping" process is observed for samples frozen in both aqueous buffer and glycerol/buffer and exhibits power law behavior with a very weak temperature dependence. A comparison of the nonexponential rebinding kinetics of CO molecules from the pumped states with the rebinding observed in flash photolysis experiments suggests that the pumped states are distinct geminate states, not observed in flash photolysis experiments. Thus, a four-state model, with two geminate states, is implicated for MbCO. Pumped states may represent "separated geminate pair" states with the CO molecule still in the heme pocket or possibly trapped within a cavity on its way through the protein matrix, consistent with molecular dynamics simulations. The possibility of significant deoxyheme relaxation from a less domed to a more domed configuration, as a result of the multiple photolysis events associated with the pumping process, is also explored. However, the small changes observed in the Soret band line shape and position subsequent to pumping at T less than 180 K tend to rule out this explanation for the pumping process. Since the yield for creating a pumped state is small (e.g., less than 10(-7) for T greater than 100 K), pumping can be observed only after extended illumination and is absent in flash photolysis measurements, even after multiple flashes. At higher temperatures (T greater than 180 K), the escape of the CO molecule to the solvent is observed. Our data are consistent with a "phase transition" of the protein that is coupled to the surrounding matrix. The protein fluctuations are quenched below approximately 185 K for a solvent composed of 70% glycerol and below approximately 260 K for aqueous buffer. We also present the first large amplitude measurements of CO rebinding from the protein exterior, observed below 200 K after freezing the sample under laser illumination.

Animals↗

PubMed1991

Spectroscopic studies of myoglobin at low pH: heme structure and ligation.

We explore heme structure and ligation subsequent to a low-pH conformational transition in sperm whale myoglobin. Below pH 4.0, the iron-histidine bond breaks in metMb and deoxyMb. In MbCO, the majority of the iron-histidine bonds remain intact down to pH 2.6; however, the observation of a weak Fe-CO mode at 526 cm-1 indicates that a small fraction of the sample has the histidine replaced by a weak ligand, possibly water. The existence of a sterically hindered CO subpopulation in MbCO and the continued association of the four-coordinate heme with the protein in deoxyMb suggest that the heme pocket remains at least partially intact in the acid-induced conformation. The global pH-dependent conformational change described here is clearly distinguished from the local "closed" to "open" transition described previously in MbCO [Morikis et al. (1989) Biochemistry 28, 4791-4800]. Further observations of the four-coordinate heme state yield insights on the mechanism of heme photoreduction and the assignment of the 760-nm band in deoxyMb.

Animals↗

PubMed1991

Spectroscopic studies of myoglobin at low pH: heme ligation kinetics.

On the basis of the characterization of heme structure and ligation in equilibrium, we explore both proximal and distal ligation kinetics of myoglobin below pH 4. Upon photolysis of MbCO, a significant five-coordinate heme population is observed, with an intact iron-histidine bond that persists on the time scale of CO rebinding. Incomplete CO photolysis is attributed to a rapidly exchanging minority population of four-coordinate hemes, which leads to fast (greater than 10(10) s-1) geminate recombination. The possible relevance of such a mechanism at pH 7 is also noted. Using a novel experimental protocol, we observe the resonance Raman spectrum of partially photolyzed MbCO as a function of continuous wave illumination time (tau). Under extended illumination (tau approximately 35 ms at pH 3.4), there is a loss of intensity in the nu 4 region of the Raman spectrum and the iron-histidine mode is bleached from the spectrum of the five-coordinate photoproduct. In the Fe-CO stretching region of the CO-bound fraction, the intensity of the 526-cm-1 mode increases with tau at the expense of the 491-cm-1 mode. These changes are interpreted as being due to replacement of the proximal histidine ligand under continuous illumination. Complete relaxation to the pure four-coordinate deoxy heme structure observed in equilibrium is not observed even as tau----infinity, presumably since CO rebinding leads to acidification of the iron and its complexation with histidine. We propose a kinetic model to account for our results and discuss the implications for previous low-pH kinetics measurements.

Animals↗