The effect of water on the rate of conformational change in protein allostery.

The influence of solvation on the rate of quaternary structural change is investigated in human hemoglobin, an allosteric protein in which reduced water activity destabilizes the R state relative to T. Nanosecond absorption spectroscopy of the heme Soret band was used to monitor protein relaxation after photodissociation of aqueous HbCO complex under osmotic stress induced by the nonbinding cosolute poly(ethylene glycol) (PEG). Photolysis data were analyzed globally for six exponential time constants and amplitudes as a function of osmotic stress and viscosity. Increases in time constants associated with geminate rebinding, tertiary relaxation, and quaternary relaxation were observed in the presence of PEG, along with a decrease in the fraction of hemes rebinding CO with the slow rate constant characteristic of the T state. An analysis of these results along with those obtained by others for small cosolutes showed that both osmotic stress and solvent viscosity are important determinants of the microscopic R --> T rate constant. The size and direction of the osmotic stress effect suggests that at least nine additional water molecules are required to solvate the allosteric transition state relative to the R-state hydration, implying that the transition state has a greater solvent-exposed area than either end state.

Absolute kinetic characterization of 17-beta-estradiol as a radical-scavenging, antioxidant synergist.

We directly measured the absolute reactivity of 17-beta-estradiol (E2) and several phenolic model compounds for E2 toward t-butoxy radical (t-BuO*) by nanosecond time-resolved optical spectroscopy. Compared to other phenols, E2 is a moderate, but not strong deactivator of oxyradicals. The absolute bimolecular rate constant for H-atom transfer from E2 to t-BuO* is 1.3 +/- 0.3 x 10(9) M(-1) x s(-1) (23 degrees C, benzene). We estimate the O-H bond strength of 17-beta-estradiol to be approximately 85 +/- 2 kcal/mol and calculate the reaction rate constant of E2 toward peroxy radical to be 10(5) M(-1) x s(-1) at 37 degrees C. The conjugate phenoxy radical of 17-beta-estradiol, E2O*, is unusually reactive toward alpha-tocopherol and ascorbate by H-atom transfer in homogeneous solution (10(8)-10(9) M(-1) x s(-1)). Our findings suggest that E2 functions in vivo as a highly localized, synergistic biological antioxidant. This may partly explain the clinical effectiveness of ovarian steroids in delaying the manifestations of Alzheimer's Disease as well as in protecting against cardiovascular pathologies. In the absence of complementary antioxidant synergists, E2O* is expected to be a pro-oxidant.

Near-ultraviolet magnetic circular dichroism spectroscopy of protein conformational states: correlation of tryptophan band position and intensity with hemoglobin allostery.

The near-UV magnetic circular dichroism spectroscopy of the aromatic amino acid bands of hemoglobin was investigated as a potential probe of structural changes at the alpha(1)beta(2) interface during the allosteric transition. Allosteric effectors were used to direct carp and chemically modified human hemoglobins into the R (relaxed) or T (tense) state in order to determine the heme-ligation-independent spectral characteristics of the quaternary states. The tryptophan magnetic circular dichroism (MCD) peak observed at 293 nm in the R state of N-ethylsuccinimide- (NES-) des-Arg-modified human hemoglobin (Hb) was shifted to a slightly longer wavelength in the T state, consistent with the shift expected for tryptophan acting as a proton donor in a T-state hydrogen bond. Moreover, the increase observed in the T-state MCD intensity of this band relative to the R-state intensity was consistent with the effect expected for proton donation by tryptophan on the basis of the Michl perimeter model of aromatic MCD. The peak-to-trough magnitude of the R - T MCD difference spectrum is equal to 30% of the total R-state peak intensity contributed by all six tryptophans present in the human tetramer; the relative magnitude specific to the two beta37 tryptophans undergoing conformational change is estimated accordingly to be 3 times larger. The Trp-beta37 spectral shift, about 200 cm(-)(1), is in good agreement with the shifts observed in other H-bonded proton donors and provides corroborating spectral evidence for the formation in solution of a T-state Trp beta37-Asp alpha94 hydrogen bond observed in X-ray diffraction studies of deoxyHb crystals.

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.

Characterization of equilibrium intermediates in denaturant-induced unfolding of ferrous and ferric cytochromes c using magnetic circular dichroism, circular dichroism, and optical absorption spectroscopies.

Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far-UV CD of the amide bands. Direct MCD spectral evidence is presented for bis-histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far-UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant-induced folding and unfolding involving a molten globulelike intermediate state (MG) with near-native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state.

Multiple pathways on a protein-folding energy landscape: kinetic evidence.

The funnel landscape model predicts that protein folding proceeds through multiple kinetic pathways. Experimental evidence is presented for more than one such pathway in the folding dynamics of a globular protein, cytochrome c. After photodissociation of CO from the partially denatured ferrous protein, fast time-resolved CD spectroscopy shows a submillisecond folding process that is complete in approximately 10(-6) s, concomitant with heme binding of a methionine residue. Kinetic modeling of time-resolved magnetic circular dichroism data further provides strong evidence that a 50-microseconds heme-histidine binding process proceeds in parallel with the faster pathway, implying that Met and His binding occur in different conformational ensembles of the protein, i.e., along respective ultrafast (microseconds) and fast (milliseconds) folding pathways. This kinetic heterogeneity appears to be intrinsic to the diffusional nature of early folding dynamics on the energy landscape, as opposed to the late-time heterogeneity associated with nonnative heme ligation and proline isomers in cytochrome c.

Spectroscopic evidence for nanosecond protein relaxation after photodissociation of myoglobin-CO.

Nanosecond time-resolved absorption and magnetic optical rotatory dispersion (MORD) measurements of photolyzed myoglobin-CO visible bands (500-650 nm) are presented. These measurements reveal a 400 ns process, spectrally distinct from ligand recombination, that accounts for 7% of the observed spectral evolution in the visible absorption bands and 4% in the MORD. The time-resolved MORD, more sensitive to heme coordination geometry than absorption, suggests that this process is most likely associated with protein relaxation on the distal side of the heme pocket, perhaps accompanying rehydration of the deoxymyoglobin photoproduct or accommodation of protein side chains to ligand escape.

Evidence for heme-heme excitonic coupling in the Soret circular dichroism of hemoglobin.

In order to study interdimer heme-heme electronic interactions in human hemoglobin, the Soret circular dichroism spectrum of the carboxy adduct is measured as a function of protein concentration, the spectrum at the highest concentration representing primarily that of alpha2beta2 tetramers (93%) and the lowest concentration representing primarily alphabeta dimers (68%). The tetramer-dimer difference spectrum, obtained using singular value decomposition and linear least squares fitting from a matrix of CD spectra measured at ten concentrations, is roughly conservative, with a larger negative lobe at shorter wavelengths and a peak-to-trough magnitude that is 18% of the tetramer's maximum Soret CD magnitude. It is tentatively assigned to heme-heme excitonic interactions on the basis of theoretical predictions by R. W. Woody [(1985) in Optical Properties and Structure of Tetrapyrroles (Blauer, G., and Sund, H., Eds.), pp. 239-256, Walter de Gruyter, New York].

Nanosecond time-resolved spectroscopy of biomolecular processes.

Over the past two decades, nanosecond absorption and vibrational spectroscopies have developed into powerful tools for monitoring the secondary, tertiary, and quaternary structural relaxations of biological macromolecules under near-physiological conditions of solvent and temperature. Observed through such methods, the dynamic response of a biomolecule to photoinitiated excursions from equilibrium can reveal valuable information about the structure-function relationship, information beyond that obtained from the static structures provided by X-ray crystallography, nuclear magnetic resonance spectroscopy, and other steady-state methods. Most recently, the development of ultra-sensitive polarization techniques for absorption spectroscopy has greatly enhanced the amount of time-resolved structural information that can be obtained from the broadened electronic spectra of biomolecules. This review examines nanosecond absorption, vibrational, and polarized absorption methods, and their applications to protein function and folding, emphasizing the complementary nature of information obtained from electronic and vibrational spectra measured on the nanosecond time scale.

Fast natural and magnetic circular dichroism spectroscopy.

The addition of circular or, more generally, elliptical polarization state detection to fast optical absorption spectroscopy can increase the amount of electronic and nuclear conformational information obtained about transient molecular species. To accomplish this, fast circular dichroism methods have emerged over the past decade that overcome the millisecond limit on time resolution associated with conventional modulation techniques and enable structural studies of excited states and kinetic intermediates. This article reviews techniques for time-resolved natural and magnetic circular dichroism spectroscopy covering the picosecond to millisecond time regimes and their applications, with particular emphasis on quasi-null ellipsometric techniques for nanosecond multichannel measurements of circular dichroism. Closely related quasi-null polarimetric techniques for nanosecond optical rotatory dispersion and linear dichroism measurements are also discussed.

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates.

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.

Allosteric intermediates in hemoglobin. 1. Nanosecond time-resolved circular dichroism spectroscopy.

Time-resolved circular dichroism (TRCD) studies performed on photolyzed hemoglobin-CO complex (HbCO) probe room temperature protein relaxations in Hb, including the R --> T allosteric transition. TRCD spectroscopy of photolysis intermediates in the near-UV (250-400 nm) spectral region provides a diagnostic for T-like structure at the alpha 1 beta 2 interface via the effect of quaternary structure on the UV CD of aromatic residues. The TRCD of porphyrin-based transitions in the UV and Soret regions, reflecting transition-dipole couplings between hemes and aromatic residues over a radius wide enough to permit heme-interface and inter-dimer interactions, is modulated by the tertiary and quaternary structure of photolysis intermediates. In the allosteric core model of Hb cooperativity, Fe-CO bond breakage initiates a heme structural change, thought to be heme doming, that is transmitted to the alpha 1 beta 2 interface via the F helix. The TRCD results, analyzed in light of kinetic information from time-resolved absorption studies, suggest specific features for the mechanism by which the ensuing tertiary and quaternary conformational changes propagate through the protein. In particular, the UV-TRCD indicates that the alpha 1 beta 2 interface responds within several hundred nanoseconds to initial events at the heme by shifting from an R toward a T-like interface. The appearance of T-like character at the alpha 1 beta 2 interface tens of microseconds before the appearance of equilibrated T state deoxyHb indicates that the R --> T transition in photolyzed HbCO is a stepwise process, as previously suggested by time-resolved resonance Raman studies.

Allosteric intermediates in hemoglobin. 2. Kinetic modeling of HbCO photolysis.

Nanosecond absorption spectra are measured in the Soret and near-UV spectral regions of human hemoglobin (Hb) after laser photolysis of the carbonyl adduct in order to study the dynamics of globin tertiary and quaternary conformational changes. Spectra and concentrations of physical intermediates, distinguished by extent of heme ligation and intraprotein relaxation, are obtained from a global analysis using a microscopic kinetic model that explicitly accounts for six observed relaxation and recombination processes. Three observed rate constants for CO rebinding and two intraprotein relaxation constants obtained are similar to constants determined by Hofrichter et al. [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2235], the latter two comprising the 20-30-microseconds R --> T quaternary transition and a previously unassigned 1-microseconds intraprotein relaxation. On the basis of the modeled intermediate spectra, as well as UV circular dichroism results observed on this time scale [Björling, S.C., Goldbeck, R.A., Paquette, S.J., Milder, S.J., & Kliger, D.S. (1996) Biochemistry 35, 8619-8627], the 1-microsecond relaxation is assigned to heme conformational changes concomitant with a relaxation of protein conformation at the alpha 1 beta 2 interface corresponding to an initial step in a compound R --> T reaction path.

A study of the mechanisms of slow religation to sickle cell hemoglobin polymers following laser photolysis.

Time-resolved linear dichroism (TRLD) measurements are conducted on gels of sickle cell hemoglobin following laser photolysis of the carbonyl adduct to monitor religation kinetics to hemoglobin S polymers. The return of the polymer phase to its equilibrium ligation state has been found to be about 1000 times slower than that of the solution phase hemoglobin tetramers. Several mechanisms describing this slow religation to the polymer were proposed: (1) religation occurs through a biomolecular process involving all polymer hemes, (2) religation occurs through a bimolecular process in which only hemoglobin molecules at the polymer ends can participate, and (3) religation occurs through the exchange of ligated hemoglobin molecules in the monomer phase with unligated ones in the polymer phase. To test these mechanisms, measurements are performed on gels having different domain sizes. The results show no relation between domain size and religation kinetics. The independence of religation kinetics and domain size is most consistent with the first of the three mechanisms described above (bimolecular recombination involving all polymer hemes). This result is discussed in terms of a model in which diffusion of the ligand is inhibited in the polymer phase. An understanding of the ligand binding kinetics of sickle hemoglobin polymers could have pathophysiological significance in its relevance to polymer formation and melting during red blood cell circulation.

Carbon monoxide religation kinetics to hemoglobin S polymers following ligand photolysis.

The re-equilibration rate of carbon monoxide binding to hemoglobin S polymers is determined by time-resolved measurements of linear dichroism spectra. Linear dichroism is used to detect religation to hemoglobin in the polymer in the presence of rebinding to free hemoglobin S tetramers. Measurement of the linear dichroism resulting from photolysis of the small percentage of ligand bound to the polymer is accomplished through the use of an ultrasensitive, ellipsometric linear dichroism technique developed for this purpose. The major finding is that the return of the polymer phase to its equilibrium ligation state is much slower than that of the solution phase hemoglobin tetramers. Assuming all hemes in the polymer are equally likely to participate in rebinding, the re-equilibration rate for carbon monoxide religation to hemoglobin S polymers is found to be 0.14 +/- 0.07 (s-1 mM-1), about 1000 times slower than the rebinding rate of carbon monoxide to T-state monomer hemoglobin. Several interpretations of this result are discussed. An understanding of the ligand binding kinetics to hemoglobin S polymers could have pathophysiological significance in its relevance to polymer formation and melting during red blood cell circulation.

Nanosecond optical rotatory dispersion spectroscopy: application to photolyzed hemoglobin-CO kinetics.

A standard technique for static optical rotatory dispersion (ORD) measurements is adapted to the measurement of ORD changes on a nanosecond (ns) time scale, giving approximately a million-fold improvement in time-resolution over conventional instrumentation. The technique described here is similar in principle to a technique recently developed for ns time-resolved circular dichroism (TRCD) spectroscopy, although the time-resolved optical rotatory dispersion (TRORD) technique requires fewer optical components. As with static ORD, TRORD measurements may be interpreted by empirical comparisons or may be transformed, via the Kramers-Kronig relations, to more easily interpreted TRCD spectra. TRORD can offer experimental advantages over TRCD in studying kinetic processes effecting changes in the chiral structures of biological molecules. In particular, the wider dispersion of ORD bands compared with the corresponding CD bands means that ORD information may often be obtained outside of absorption bands, a signal-to-noise advantage for multichannel measurements. Demonstration of the technique by its application to ns TRORD and the transform-calculated TRCD of carboxy-hemoglobin (Hb-CO) after laser photolysis is presented.

Nanosecond absorption study of kinetics associated with carbon monoxide rebinding to hemoglobin S and hemoglobin C following ligand photolysis.

The absorption spectra of photolysis intermediates of the CO complex of hemoglobin S and hemoglobin C, in the tetramer form, have been measured between 10 ns and 200 ms after excitation. These data were analyzed using singular value decomposition (SVD) and global analysis to determine kinetic lifetimes associated with various processes involved in CO recombination. The results of this analysis show that, in the tetramer (non-aggregated) form, hemoglobin S and hemoglobin C exhibit the same kinetics associated with CO recombination as hemoglobin A.

Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio.

The UV-visible absorption and magnetic circular dichroism (MCD) spectra of the ferric, ferrous, CO-ligated forms and kinetic photolysis intermediates of the tetraheme electron-transfer protein cytochrome c3 (Cc3) are reported. Consistent with bis-histidinyl axial coordination of the hemes in this Class III c-type cytochrome, the Soret and visible region MCD spectra of ferric and ferrous Cc3 are very similar to those of other bis-histidine axially coordinated hemeproteins such as cytochrome b5. The MCD spectra indicate low spin state for both the ferric (S = 1/2) and ferrous (S = 0) oxidation states. CO replaces histidine as the axial sixth ligand at each heme site, forming a low-spin complex with an MCD spectrum similar to that of myoglobin-CO. Photodissociation of Cc3-CO (observed photolysis yield = 30%) produces a transient five-coordinate, high-spin (S = 2) species with an MCD spectrum similar to deoxymyoglobin. The recombination kinetics of CO with heme Fe are complex and appear to involve at least five first-order or pseudo first-order rate processes, corresponding to time constants of 5.7 microseconds, 62 microseconds, 425 microseconds, 2.9 ms, and a time constant greater than 1 s. The observed rate constants were insensitive to variation of the actinic photon flux, suggesting noncooperative heme-CO rebinding. The growing in of an MCD signal characteristic of bis-histidine axial ligation within tens of microseconds after photodissociation shows that, although heme-CO binding is thermodynamically favored at 1 atm CO, binding of histidine to the sixth axial site competes kinetically with CO rebinding.

Solvent and temperature effects on the excited singlet state absorption of diphenylbutadiene.

Nanosecond excited state absorption spectra of all-trans-1,4-diphenyl-1,3-butadiene (DPB) and a rigid s-cis DPB analog, 1,4-diphenyl-1,3-cyclopentadiene, were obtained in several hydrocarbon solvents at room temperature and low temperatures. Analysis of the excited state absorption spectra of these two molecules suggests the presence of excited state s-cis rotamers in DPB at room temperature.