Mechanisms for excited state relaxation and dissociation of oxymyoglobin and carboxymyoglobin.

195

152

Photolysis ofHbCO, MbCO, and CO-protoheme has been investigated by measuring transient differential spectra and kinetics of induced absorption after excitation with. a 250-fsec laser pulse at 307 nm. Probing was performed by a part of a continuum pulse between 395 and 445 nm. Photodissociation of the three liganded species occurred within the pulse duration. By contrast, the formation ofdeoxy species appeared with a mean (± SD) response time of 350 ± 50 fsec. This time constant was identical for the three species and independent of the presence or absence of the protein structure. Our results suggest the formation of a transient high-spin in plane iron (II) species which relaxes in 350 fsec to a high-spin stable state with concerted kinetics of CO departure and iron displacement. The spin transition is suspected to occur via liganded excited states which relax in part to nonreactive states with a 3.2-psec time constant.Picosecond spectroscopy has revealed that the dissociation of ligand from the heme in Mb and Hb occurs within 4 psec and that the nonliganded species (Mb or Hb) were formed with a time constant ofill psec (1, 2). This has been confirmed recently by Cornelius et al. (3) who observed the appearance of Mb species after photodissociation of MbO2 with a 12-psec time constant; their results on MbCO showed a "simultaneous development of both bleaching and absorption intensities." From results of studies with 0.5-psec optical pulses at 615 nm in a pump probe experiment, Shank et al. (4) deduced that the photolysis of CO from HbCO should occur in less than 0.5 psec.More recently, Greene et al (5) and Chernoff et al. (6), using 8-psec excitation pulses at 353 and 530 nm, obtained transient absorption spectra in the Soret and visible regions at 10 and 680 psec which indicated that deoxy species formed in a time shorter than their-pulse duration. Terner et al. (7) In the present work, we have limited ourselves to the investigation of the kinetics of formation of the nonliganded species after dissociation of CO from MbCO and HbCO. In order to appreciate the possible influence of polypeptidic environment on the heme reactivity in these hemoproteins, we have also studied the kinetics ofCO dissociation from protoheme and ofdeoxyprotoheme formation. These experiments were carried out with a newly developed spectroscopic technique operating with laser pulses of250 fsec duration to excite the hemes at 307 nm. MATERIAL AND METHODS Preparation ofthe Hemoproteins and Protoheme Solutions.Purified human adult hemoglobin was prepared from fresh human blood by DEAE-Sephadex chromatography (10). The experimental solution was 0.1 mM on the basis of heme; it was diluted in 0.1 M K phosphate buffer at pH 7 or 8. The solution was first deoxygenated under moist argon before equilibration with CO at 1 atm (1 atm = 1.013 x 105 pascals). MetMb (horse heart type III, Sigma) was converted to MbFe2' after addition of a 5 molar excess of freshly prepared Na dithionite (Merck) under strict anaerobic conditions and then equil...

supporting

confidence: 70%

mentioning

confidence: 99%

Femtosecond photolysis of CO-ligated protoheme and hemoproteins: appearance of deoxy species with a 350-fsec time constant.

Martin¹,

Migus²,

Poyart³

et al. 1983

195

152

“…These calculations fit well the spectra observed below 300 K. The parameters allow calculations of the differences between spectra at 600 K and 300 K that quantify the following expected features: peaks at ±13 nm and a diminution of -20% in peak intensity. Such effects would directly influence the observed heme protein transient absorption spectra (1)(2)(3)(4)(5)(6)(7)(8)(9). Studies on absorption transients of iron porphyrins (38,39) show spectra that contain features like those expected for heating, namely an apparent bleaching of the Soret band and the appearance of positive optical density change on the wings of the absorption band.…”

mentioning

confidence: 99%

Molecular dynamics simulations of cooling in laser-excited heme proteins.

Henry¹,

Eaton²,

Hochstrasser³

1986

274

237

In transient optical experiments the absorbed photon raises the vibrational temperature of the chromophore. In heme proteins at room temperature conversion of a 530-nm photon into vibrational energy is estimated to raise the temperature of the heme by 500-700 K. Cooling of the heme is expected to occur mainly by interacting with the surrounding protein. We report molecular dynamics simulations for myoglobin and cytochrome c in vacuo that predict that this cooling occurs on the ps time scale. The decay of the vibrational temperature is nonexponential with about 50% loss occurring in 1-4 ps and with the remainder in 20-40 ps. These results predict the presence of nonequilibrium vibrational populations that would introduce ambiguity into the interpretation of transient ps absorption and Raman spectra and influence the kinetics of sub-ns geminate recombination.slowly than the time resolution of conventional ps laser experiments (12-15). There is, therefore, a reasonable expectation that the heme group in the protein will retain vibrational energy in excess of equilibrium for measurably long times following optical excitation.If one or more photons of wavelength X were absorbed by a heme group that was initially at equilibrium at 300 K with average vibrational energy (E) exclusive of zero-point energy and if the excitation were distributed by the intramolecular vibrational relaxation over all the harmonic vibrational modes of the ground electronic state in accordance with Boltzmann statistics, the new molecular temperature T would be obtained by satisfying the relationAfter molecules absorb visible or ultraviolet light, the deposited energy becomes distributed over a subset of the molecular motions and subsequently is exchanged with the surroundings. When ps or fs light pulses are used to probe photophysical or photochemical processes, it is necessary to consider that nonequilibrium distributions of vibrational energy may persist during the time scale of the experiment. Transient excesses of vibrational energy can alter the rates of chemical and physical transformations, and they may also introduce spectral changes that could easily be mistaken for chemical steps in a process.Transient spectra of heme proteins have been studied with ps (1-7) and fs (8, 9) time resolution in experiments that have exposed a variety of spectral intermediates following light absorption by the heme group. The principal goal of such experiments is to understand the sequence of early structural alterations occurring after a diatomic ligand (02, CO, NO) is photodissociated from the heme iron. On these time scales the photolyzed sample may also contain electronically excited states, each of which may have a spectrum that changes with time as a result of changes in its vibrational energy content. The present paper is an attempt to predict, on the basis of molecular dynamics simulations, the rate at which the excess vibrational energy in the heme is dissipated into the protein.To understand the detailed pathways for vibrational cooling ...

“…Myoglobin has a similar electronic structure to hemoglobin but small spectral shifts result from the differences in protein environment. Because of its resemblance to a single hemoglobin subunit, myoglobin provides a natural model for the tetrameric protein, and it has been the object of previous kinetic studies in both picosecond (3,4) and longer time regimes (5, 6). The spectral and temporal information provided by our picosecond spectrometer makes possible a more detailed investigation of the primary photolytic processes in these molecules.…”

mentioning

confidence: 99%

Different dissociation pathways and observation of an excited deoxy state in picosecond photolysis of oxy- and carboxymyoglobin.

Cornelius¹,

Steele²,

Chernoff³

et al. 1981

Picosecond transient absorption spectra of Mb, MbCO, and MbO2 have been studied at time delays.of up to 10 ns after excitation at 353 nm. Particular attention has been paid to the rapid spectral changes that occur in the Soret region during the first 50 ps in MbCO and MbO2. In MbCO both the bleaching of the Soret peak (feature I) and the appearance of new deoxy-like absorption (feature II) occur instantaneously, whereas in MbO2 feature II is delayed with respect to feature I. A short-lived ('=12 ps) feature near 455 nm (feature III) was much more intense in MbO2 than in MbCO and was also identified in the transient spectrum of Mb. No evidence of subnanosecond geminate recombination was found in either MbCO or MbO2. These observations are consistent with a scheme in which MbO2 photodissociates through an excited state of Mb, whereas MbCO under the same conditions produces ground state Mb directly. The results and conclusions are compared with those of previous picosecond studies on these molecules and related hemoglobin derivatives.Picosecond transient absorption spectroscopy is a valuable method for the study ofheme proteins and their reactions with 02 and CO. The unique feature of this technique is its ability to probe changes occurring on a time scale inwhich any largescale movements of the protein are unlikely. In the case of hemoglobin, this permits the observation of the primary processes that initiate the protein conformational changes associated with cooperativity. Understanding these primary processes requires a detailed knowledge of the electronic states of this system and the couplings and interactions between them. To this end, a great deal of experimental and theoretical work has been devoted to establishing an accurate mapping of the states ofvarious hemoglobin derivatives (1). We recently reported the results of a series of picosecond transient measurements on HbCO and HbO2, in which we showed that relaxation pathways derived from spin-orbit coupling arguments could be used to explain the observed spectral changes (2). That work showed how such considerations could account for the difference in the behavior ofthese two molecules: HbO2 exhibited a fast transient (<90 ps) and subnanosecond geminate recombination, whereas in HbCO both of these effects were absent.In order to examine further the scheme proposed in these hemoglobin studies, we have used our picosecond transient absorption spectrometer to study a closely related set ofmyoglobin derivatives: MbCO, MbO2, and unliganded Mb. Myoglobin has a similar electronic structure to hemoglobin but small spectral shifts result from the differences in protein environment. Because of its resemblance to a single hemoglobin subunit, myoglobin provides a natural model for the tetrameric protein, and it has been the object of previous kinetic studies in both picosecond (3,4) and longer time regimes (5, 6). The spectral and temporal information provided by our picosecond spectrometer makes possible a more detailed investigation of the primary photolyt...