Iron-carbonyl bond geometries of carboxymyoglobin and carboxyhemoglobin in solution determined by picosecond time-resolved infrared spectroscopy.

Abstract

The iron-carbonyl geometries in carboxymyoglobin (MbCO) and carboxyhemoglobin (HbCO) in ambient temperature solution have been investigated using picosecond time-resolved infrared spectroscopy. Polarized infrared and visible beams were used to monitor the change in infrared absorbance of the bound CO stretch bands on photodissociation of the ligand. The ratio of the change in absorbance for perpendicular and parallel relative polarizations of the photolysis and infrared probe beams is directly related to the angle between the ligand bond axis and the normal to the heme plane. Ratios, and hence the angles, have been obtained for the configurations giving rise to the principal CO We have recently suggested a new method of determining IR spectra with ps time resolution (2). In this measurement the sample is first pumped by a ps pulse of visible or UV light and the IR spectrum of the sample consisting of both photoproducts and unphotolyzed material can be recorded at variable ps delay times. The experiment can be done with polarized IR and visible beams, so that the magnitude and decay of the orientational correlation function for photoproducts and unphotolyzed material can be evaluated. For heme proteins-where the overall molecular rotation is slow-the magnitude of the polarization anisotropy is directly related to the angle between the transition dipoles for the IR and visible absorptions. In this paper we present results from a significantly improved version of the original experimental design, applied to the photodissociation of carboxymyoglobin (MbCO) and carboxyhemoglobin (HbCO). The measurements are used to define the heme-CO geometry for these heme proteins in solution. The preliminary results (2) for MbCO involved an uncertainty in the polarization anisotropy of >50%. In this study the error is reduced to -10% and allows a sharp definition of the ligand geometry of this heme protein in solution. MATERIALS AND METHODSSpectroscopic Method. The transient IR spectra were recorded using the same principles as previously described (2 The polarization experiment was carried out as follows. The upconversion signal intensity was measured both with the photolyzing beam blocked and with the photolyzing beam incident on the sample. The A/2 plate in the photolyzing beam was then rotated such that the polarization of that beam was rotated by 900, and the signal measurements were repeated.In this way, the change in IR absorbance could be measured for both parallel and perpendicular IR/photolysis laser polarizations. Measurements were taken using a time delay of 40 ps between the pump and probe lasers. A correction factor of ==5% was required to compensate for the difference in reflectance for the two polarizations of the dichroic beam Abbreviations: MbCO, carboxymyoglobin; HbCO, carboxyhemoglobin. 5062The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Perturbations of the distal heme pocket in human myoglobin mutants probed by infrared spectroscopy of bound CO: correlation with ligand binding kinetics.

“…That work demonstrates that the apparent angle a of the CO relative to the heme normal (shown in Fig. 1) in the conformers observed for SW Mb is in the inverse order of their peak frequencies (27,28).t The opposite trend would be expected if the carbon of the CO was located directly above the iron, and the observed angle a measured only the bend of the Fe-C-O from linearity. This is because a reduction in the Fe-C-O angle (increase in a) should decrease Fe-r (CO) back bonding, strengthening the CO bond and thus increasing the peak frequency.…”

“…*The interpretation of linear dichroism experiments in terms of the angle a between the CO bond direction and the heme normal tacitly assumes that the direction of the transition dipole moment for the IR band lies along the CO bond direction (27,28 (12,14). The absence of the slow rebinding A3 conformer in human Mb may explain why CO rebinding to human Mb after flash photolysis (15,16) is faster than for SW Mb (13).…”

Interactions between Heme d and Heme b595 in Quinol Oxidase bd from Escherichia coli: A Photoselection Study Using Femtosecond Spectroscopy

“…Indeed, such effects have been observed in this work, and we have exploited them to elicit the spectroscopic properties and interaction between hemes d and b 595 in more detail than was possible in a previous work (14), performed under parallel excitation conditions only. Interestingly, this is the first instance of studying photoselection effects in UV/vis transient absorption experiments of heme proteins, including the classical "model" systems such as myoglobin, presumably because such effects are not too informative for monoheme mechanisms [photoselection effects have been exploited in transient infrared spectroscopy, where they can yield structural information (32,33)]. …”

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

“…Polarization-dependent UV-pump IR-probe spectroscopy provides insight into the relative angles between the optical and IR-transition dipole moments (37,38,55). By monitoring IR signals ΔA ‖ and ΔA ⊥ when the polarizations of optical-pump and IR-probe beams are, respectively, parallel and perpendicular to each other, an anisotropy γ can be calculated which is related to the angle ϕ between the dye's optical-and the probe's IR-transition dipole moments as (42)…”

“…The solvation dynamics for the dye bound at the active site of the enzyme was found to be very different from what is observed for the same dye in water, suggesting that the active site solvating dipoles were highly restricted. In the present work, we directly measure the extent of reorganization of probe dipoles in the enzyme active site by employing polarization-and time-dependent IR spectroscopy (time-resolved UVpump IR-probe spectroscopy) (37)(38)(39), as an ultrafast probe of solvation dynamics. This spectroscopic method combines the subpicosecond time resolution of the optical laser pulses with atomic level structural sensitivity of vibrational spectroscopy.…”