Proximal and distal influences on ligand binding kinetics in microperoxidase and heme model compounds

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound heme complexes to study proximal and distal influences on ligand rebinding kinetics. We report kinetics of CO rebinding to microperoxidase (MP) and 2-methylimidazole ligated Fe protoporphyrin IX in the 10 ns to 10 ms time window. We also report CO rebinding kinetics of MP in the 150 fs to 140 ps time window. For dilute, micelle-encapsulated (monodisperse) samples of MP, we do not observe the large amplitude geminate decay at approximately 100 ps previously reported in time-resolved IR measurements on highly concentrated samples [Lim, M., Jackson, T. A., and Anfinrud, P. A. (1997) J. Biol. Inorg. Chem. 2, 531-536]. However, for high concentration aggregated samples, we do observe the large amplitude picosecond CO geminate rebinding and find that it is correlated with the absence of the iron-histidine vibrational mode in the time-resolved Raman spectrum. On the basis of these results, the energetic significance of a putative distal pocket CO docking site proposed by Lim et al. may need to be reconsidered. Finally, when high concentration samples of native myoglobin (Mb) were studied as a control, an analogous increase in the geminate rebinding kinetics was not observed. This verifies that studies of Mb under dilute conditions are applicable to the more concentrated regime found in the cellular milieu.     

Investigations of photolysis and rebinding kinetics in myoglobin using proximal ligand replacements

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound H93G myoglobin (Mb) mutants to study the influence of the proximal ligand on the CO rebinding kinetics. In H93G mutants, where the proximal linkage with the protein is eliminated and the heme can bind exogenous ligands (e.g., imidazole, 4-bromoimidazole, pyridine, or dibromopyridine), we observe significant effects on the CO rebinding kinetics in the 10 ns to 10 ms time window. Resonance Raman spectra of the various H93G Mb complexes are also presented to aid in the interpretation of the kinetic results. For CO-bound H93G(dibromopyridine), we observe a rapid large-amplitude geminate phase with a fundamental CO rebinding rate that is approximately 45 times faster than for wild-type MbCO at 293 K. The absence of an iron proximal ligand vibrational mode in the 10 ns photoproduct Raman spectrum of CO-bound H93G(dibromopyridine) supports the hypothesis that proximal ligation has a significant influence on the kinetics of diatomic ligand binding to the heme.     

Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination

Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.     

Kinetic modulation in carbonmonoxy derivatives of truncated hemoglobins: the role of distal heme pocket residues and extended apolar tunnel

Truncated hemoglobins (trHbs), are a distinct and newly characterized class of small myoglobin-like proteins that are widely distributed in bacteria, unicellular eukaryotes, and higher plants. Notable and distinctive features associated with trHbs include a hydrogen-bonding network within the distal heme pocket and a long apolar tunnel linking the external solvent to the distal heme pocket. The present work compares the geminate and solvent phase rebinding kinetics from two trHbs, one from the ciliated protozoan Paramecium caudatum (P-trHb) and the other from the green alga Chlamydomonas eugametos (C-trHb). Unusual kinetic patterns are observed including indications of ultrafast (picosecond) geminate rebinding of CO to C-trHb, very fast solvent phase rebinding of CO for both trHbs, time-dependent biphasic CO rebinding kinetics for P-trHb at low CO partial pressures, and for P-trHb, an increase in the geminate yield from a few percent to nearly 100% under high viscosity conditions. Species-specific differences in both the 8-ns photodissociation quantum yield and the rebinding kinetics, point to a pivotal functional role for the E11 residue. The response of the rebinding kinetics to temperature, ligand concentration, and viscosity (glycerol, trehalose) and the viscosity-dependent changes in the resonance Raman spectrum of the liganded photoproduct, together implicate both the apolar tunnel and the static and dynamic properties of the hydrogen-bonding network within the distal heme pocket in generating the unusual kinetic patterns observed for these trHbs.     

T-state hemoglobin with four ligands bound

Flash photolysis kinetics have been measured for ligand recombination to hemoglobin (Hb) in the presence of two effectors: bezafibrate (Bzf) and inositol hexakisphosphate (IHP). The combined influence of the two independent effectors leads to predominantly T-state behavior. Samples equilibrated with 0.1 atm of CO are fully saturated, yet after photodissociation they show only T-state bimolecular recombination rates at all photolysis levels; this indicates that the allosteric transition from R to T occurs before CO rebinding and that the allosteric equilibrium favors the T-state tetramer with up to three ligands bound. Since all four ligands bind at the rate characteristic for the T-state, the return transition from T to R must occur after the fourth ligand was bound. At 1 atm of CO, rebinding to the initial R state competes with the allosteric transition resulting in a certain fraction of CO bound at the rate characteristic for the R state; this fraction is greater the smaller the percentage dissociation. Under 1 atm of oxygen, samples are not more than 93% saturated and show mainly T-state kinetics. The results show that all four hemes can bind oxygen or CO ligands in the T structure. The fraction of the kinetics occurring as geminate is less for partially liganded (T-state) samples than for fully liganded (R-state) Hb.     

Ligand binding dynamics to the heme domain of the oxygen sensor Dos from Escherichia coli

In the heme-based oxygen sensor Dos from Escherichia coli, one of the axial ligands (Met 95) of a six-coordinate heme can be replaced by external ligands such as O(2), NO, and CO, which causes a switch in phosphodiesterase activity. To gain insight into the bidirectional switching mechanism, we have studied the interaction of ligands with the sensor domain DosH by flash photolysis experiments with femtosecond time resolution. The internal ligand can be photodissociated from the ferrous heme and recombines with time constants of 7 and 35 ps. This is somewhat slower than recombination of the external ligands NO, with which picosecond rebinding occurs with unprecedented efficiency (>99%) with a predominant phase of approximately 5 ps, and O(2) (97% in 5 ps, Liebl, U., Bouzhir-Sima, L., Négrerie, M., Martin, J.-L., and Vos, M. H. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 12771-12776). Dissociated CO displays geminate rebinding in 1.5 ns with a very high yield (60%). Together these results indicate that the heme environment provides a very tight pocket for external ligands, presumably preventing frequent switching events. Additional CO dissociation and rebinding experiments on a longer time scale reveal that (a) Met 95 binding, in 100 micros, occurs in competition with bimolecular CO binding, and (b) subsequent replacement of Met 95 by CO on the millisecond time scale occurs faster than in rapid-mixing experiments, suggesting a slow further relaxation. A minimal ligand binding model is proposed that suggests that Met 95 displacement from the heme is facilitated by the presence of an external ligand in the heme environment. Furthermore, the orders of magnitude difference between Met 95 binding after dissociation of internal and external ligands, as well as the spectral characteristics of photodissociation intermediates, indicate substantial rearrangement of the heme environment associated with ligand sensing. Further remarkable observations include evidence for stable (>4 ns) photooxidation of six-coordinate ferrous heme, with a quantum yield of 4-8%.     

Ligand binding to a hemoprotein lacking the distal histidine. The myoglobin from aplysia limacina (Val(E7))

The time course of ligand recombination to the myoglobin from Aplysia limacina, which has Val(E7), was measured following photolysis by flashes of 35 ps to 300 ns with a time resolution of 10 ps or 1 ns. CO shows only biomolecular recombination. O2 has a small geminate reaction with a half-time of tens of picoseconds, but no nanosecond geminate reaction. NO has two picosecond relaxations with half-times of 70 ps (15%) and 1 ns (80%) and one nanosecond relaxation with a half-time of 4.6 ns. The biomolecular rates for O2 and NO are the same: 2 x 10(7) M-1 s-1. Methyl and ethyl isonitriles have a geminate reaction with a half-time of 35 ps. Ethyl isonitrile has, in addition, a nanosecond relaxation (25%) with a half-time of 100 ns. t-Butyl isonitrile has four geminate relaxations (10 ps, 35 ps, 1 ns, and 1 microseconds). Analysis of the results suggests much easier movement of ligand between the heme pocket and the exterior than in sperm whale myoglobin (His(E7]. The reactivity of the heme is little different, placing the effect of the differences from sperm whale myoglobin on the distal side of the heme.     

Multiple geminate ligand recombinations in human hemoglobin

The geminate ligand recombination reactions of photolyzed carbonmonoxyhemoglobin were studied in a nanosecond double-excitation-pulse time-resolved absorption experiment. The second laser pulse, delayed by intervals as long as 400 ns after the first, provided a measure of the geminate kinetics by rephotolyzing ligands that have recombined during the delay time. The peak-to-trough magnitude of the Soret band photolysis difference spectrum measured as a function of the delay between excitation pulses showed that the room temperature kinetics of geminate recombination in adult human hemoglobin are best described by two exponential processes, with lifetimes of 36 and 162 ns. The relative amounts of bimolecular recombination to T- and R-state hemoglobins and the temperature dependence of the submicrosecond kinetics between 283 and 323 K are also consistent with biexponential kinetics for geminate recombination. These results are discussed in terms of two models: geminate recombination kinetics modulated by concurrent protein relaxation and heterogeneous kinetics arising from alpha and beta chain differences.     

Flash-photolysis of fully reduced and mixed-valence CO-bound Rhodobacter sphaeroides cytochrome c oxidase: heme spectral shifts

Conformational changes, internal electron transfer, and CO rebinding processes in cytochrome c oxidase from Rhodobacter sphaeroides reduced to different degrees were investigated. The reactions were followed using a gated optical spectrometric multichannel analyzer. Light-induced difference spectra, recorded in the 350-700 nm region over the 100 ns to 1 s time interval, were analyzed by singular value decomposition and global exponential fitting. The photolyzed fully reduced enzyme showed two relaxations, approximately 1 and 190 mus, prior to the 20 ms CO rebinding process. Intramolecular electron transfer was monitored following photolysis of the mixed-valence CO-bound enzyme. The analysis revealed 1.1 micros, 2.4 micros, 31 micros, 68 ms, and 240 ms apparent lifetimes, the first three of which are attributed to electron transfer from heme a3 to heme a with contribution from a relaxation process at the heme a3 site. Spectral changes associated with the microsecond processes are consistent with 75% electron transfer from heme a3 to heme a. A comparison of the experimental spectra and model difference spectra for the intramolecular electron transfer indicated approximately 3 nm blue shift in the absolute spectra of both the oxidized heme a3 and reduced heme a generated in the process. The 68 and 240 ms lifetimes are due to CO recombination to heme a3 and are attributed to the presence of two conformers, the slower rate corresponding to the conformer in higher abundance. The dependency of the apparent rate of CO rebinding on the intensity of the probe beam in single-wavelength experiments is explained.     

Different roles of protein dynamics and ligand migration in non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana

The ligand rebinding kinetics after photolysis of the CO complexes of Arabidopsis thaliana hemoglobins AHb1 and AHb2 in solution show very different amplitudes in the geminate phase, reflecting different migration pathways of the photodissociated ligand in the system of internal cavities accessible from the heme. The dependence of the geminate phase on CO concentration, temperature, encapsulation in silica gels and presence of glycerol confirms a remarkable difference in the internal structure of the two proteins and a dramatically different role of protein dynamics in regulating the reactivity with CO. This finding strongly supports the idea that they have distinct physiological functions.     

Dynamics and reactivity of HbXL99 alpha. A cross-linked hemoglobin derivative

Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.     

Myoglobin mutants giving the largest geminate yield in CO rebinding in the nanosecond time domain.

We have measured the rebinding of carbon monoxide (CO) to some distal mutants of myoglobin (Mb) in the time range from 10(-8) to 10(-1) s by flash photolysis, in which the photodissociated CO rebinds to the heme iron without escaping to the solvent water from the protein matrix. We have found that the double mutants [His64-->Val/Val68-->Thr (H64V/V68T) and His64-->Val/Val68-->Ser (H64V/V68S)] have an extremely large geminate yield (70-80%) in water at 5 degreesC, in contrast to the 7% of the geminate yield of wild-type Mb. The CO geminate yields for these two mutants are the largest in those of Mb mutants reported so far, showing that the two mutants have a unique heme environment that favors CO geminate rebinding. Comparing the crystal structures and 1H-NMR and vibrational spectral data of H64V/V68T and H64V/V68S with those of other mutants, we discuss factors that may control the nanosecond geminate CO rebinding and CO migration in the protein matrix.     

The relation of ligand binding to redox state in the hexa-heme nitrite reductase of Wolinella succinogenes.

Ligand binding reactions and the relation between redox state and ligand binding in the hexa-heme nitrite reductase of Wolinella succinogenes have been studied using laser flash photolysis. On a picosecond time scale, a rapid excursion was observed corresponding to the breaking and reforming of an iron histidine bond. With the CO derivative, a geminate reaction was observed with a rate of 3 ns-1. On a nanosecond time scale, no slower geminate reactions were observed. For the cyanide derivative, no geminate reactions were observed at either time scale. The second order reaction of CO with the enzyme had a time course consisting of two distinct components. This time course changed in form as the enzyme came to equilibrium with CO, and the slower rebinding component was replaced by a faster rebinding component. It is suggested that CO binding enhances reduction of a heme with an unusually low redox potential and opens the structure of the active site to allow a faster second order reaction of CO. The proportion of the geminate CO reaction was unchanged, consistent with changes relatively remote from the ligand binding site. The second order reactions of cyanide also showed that redox effects influence its rebinding reaction. Adding cyanide to the CO complex of nitrite reductase showed that the two ligands have distinct heme binding sites.     

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quaternary structure dependence of kinetic hole burning and conformational substates interconversion in hemoglobin

Using a sol-gel encapsulation technique, we have prepared samples of CO saturated human adult hemoglobin locked in the R or T quaternary conformation. We report time-resolved spectra of these samples in the Soret region following flash photolysis, in the time interval ranging from 250 ns to 200 ms and in the temperature interval of 100-170 K. A suitable analysis of the measured difference spectra enables us to obtain the spectral contribution of deoxyHb and HbCO molecules as a function of time and/or of the fraction N(t) of deoxyHb molecules. In our experimental time window geminate CO rebinding to hemoglobin in the T quaternary conformation is about 2 orders of magnitude slower than to hemoglobin in the R conformation: this suggests that the barrier distribution for the CO rebinding, g(H), depends strongly on the protein quaternary structure. In our temperature interval, spectral shifts due to kinetic hole burning (KHB) are present: for HbCO the KHB effect is large in the R conformation and small in the T conformation. For deoxyHb the opposite is true. We attribute the observed behavior to the effect of interconversion between the relevant substates. This effect is stronger for HbCO molecules in the T conformation and for deoxyHb molecules in the R conformation; it confirms the quaternary structure dependence of the hemoglobin energy landscape and suggests enhanced dynamics of ligation intermediate species such as T-state HbCO or R-state deoxyHb.     

Ligand dynamics in an electron transfer protein. Picosecond geminate recombination of carbon monoxide to heme in mutant forms of cytochrome c

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to approximately 70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.     

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.     

Dynamics of NO rebinding to the heme domain of NO synthase-like proteins from bacterial pathogens

Some Gram-positive bacterial pathogens harbor a gene that encodes a protein (HNS, Heme domain of NO Synthase-like proteins) with striking sequence identity to the oxygenase domain of mammalian NO synthases (NOS). However, they lack the N-terminal and the Zn-cysteine motif participating to the stability of an active dimer in the mammalian isoforms. The unique properties of HNS make it an excellent model system for probing how the heme environment tunes NO dynamics and for comparing it to the endothelial NO synthase heme domain (eNOS(HD)) using ultrafast transient spectroscopy. NO rebinding in HNS from Staphylococcus aureus (SA-HNS) is faster than that measured for either Bacillus anthracis (BA-HNS) or for eNOS(HD) in both oxidized and reduced forms in the presence of arginine. To test whether these distinct rates arise from different energy barriers for NO recombination, we measured rebinding kinetics at several temperatures. Our data are consistent with different barriers for NO recombination in SA-HNS and BA-HNS and the presence of a second NO-binding site. The hypothesis that an additional NO-binding cavity is present in BA-HNS is also consistent with the effect of the NO concentration on its rebinding. The lack of the effect of NO concentration on the geminate rebinding in SA-HNS could be due to an isolated second site. We confirm the existence of a second NO site in the oxygenase domain of the reduced eNOS as previously hypothesized [A. Slama-Schwok, M. Négrerie, V. Berka, J.C. Lambry, A.L. Tsai, M.H. Vos, J.L. Martin, Nitric oxide (NO) traffic in endothelial NO synthase. Evidence for a new NO binding site dependent on tetrahydrobiopterin? J. Biol. Chem. 277 (2002) 7581-7586]. This site requires the presence of arginine and BH(4); and we propose that NO dynamic and escape from eNOS is regulated by the active site H-bonding network connecting between the heme, the substrate, and cofactor.