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

Abstract

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...

Spin-dependent mechanism for diatomic ligand binding to heme

“…The calculations of Im-FeP-XO (X ϭ C, N, and O) models consider geometries at Q P ϭ 0.2 and 0.4 Å to include two possible out-of-plane geometries. Given the rapidity of photolysis, it is reasonable to assume that the spin state of the iron is randomized relative to that of the photolyzed ligand (2,50). The assumption that the spin states of the free ligands and the iron are uncorrelated implies that possible spin states are S Fe ϩ S ligand , S Fe ϩ S ligand Ϫ 1, .…”

Picosecond Dynamics of CN--Ligated Ferric Cytochrome c after Photoexcitation Using Time-resolved Vibrational Spectroscopy

“…[1][2][3][4] The binding of a small ligand to heme proteins has been greatly studied in order to understand the relationships between protein structure, dynamics, and function. 2,3,[5][6][7][8][9][10][11][12][13][14] In particular, the rebinding dynamics of a number of neutral diatomic molecules, such as CO, NO, or O 2 to ferrous heme or heme proteins has been widely investigated both experimentally and theoretically. By contrast, only a few experiments have been carried out on the rebinding dynamics of ionic molecules to heme proteins.…”

Functional consequences of mutations at the allosteric interface in hetero‐ and homo‐hemoglobin tetramers

“…They showed that 35 ps after the light pulse, a transient spectrum was observed with a band maximum located at 765 nm, i.e., red shifted by almost 6 nm relative to the deoxy HbA band 111. This transient remained unchanged up to 60 ns, indicating that the geometry of the heme group is only partly relaxed and that equilibration of the surrounding protein structure occurs on a longer time scale (Sawicki &Gibson, 1976;Martin et al, 1983;Petrich et al, 1991). Figure 2 shows the equilibrium characteristics of band I11 in the deoxy HbA (maximum absorbance at 760 nm) compared to that of the deoxy p4 tetramer, which is red shifted infrared by almost 6 nm, i.e., at nearly the same wavelength as the 35 ps transient reported by Dunn and Simon (1991) after photodissociation of CO HbA.…”