Time-resolved Magnetic Circular Dichroism Research Articles

Magnet-Free Time-Resolved Magnetic Circular Dichroism with Pulsed Vector Beams.

Magnetic circular dichroism (MCD) is a widely used spectroscopic technique which reveals valuable information about molecular geometry and electronic structure. However, the weak signal and the necessary strong magnets impose major limitations on its application. We propose a novel protocol to overcome these limitations by using pulsed vector beams (VBs), which consist of nanosecond gigahertz pump and femtosecond UV-vis probe pulses. By virtue of the strong longitudinal electromagnetic fields, the MCD signal detected by using the pulsed VBs is greatly enhanced compared to conventional MCD performed with plane waves. Furthermore, varying the pump-probe time delay allows monitoring the ultrafast variation of molecular properties.

Light-wave dynamic control of magnetism.

The enigmatic interplaybetween electronic and magnetic phenomena observed in manyearly experiments and outlinedin Maxwell's equations propelled the development of modern electromagnetism1. Today, the fully controlled evolution of the electric field of ultrashort laser pulses enables the direct and ultrafast tuning of the electronic properties of matter, which is the cornerstone of light-wave electronics2-7. By contrast, owing to the lack of first-order interaction between light and spin, the magnetic properties of matter can only be affected indirectly and on much longer timescales, through a sequence of optical excitations and subsequent rearrangement of the spin structure8-16. Here we introduce the regime of ultrafast coherent magnetism and show how the magnetic properties of a ferromagnetic layer stack can be manipulated directly by the electric-field oscillations of light, reducing the magnetic response time to an external stimulus by two orders of magnitude. To track the unfolding dynamics in real time, we develop an attosecond time-resolved magnetic circular dichroism detection scheme, revealing optically induced spin and orbital momentum transfer in synchrony with light-field-driven coherent charge relocation17. In tandem with ab initio quantum dynamical modelling, we show how this mechanism enables the simultaneous control of electronic and magnetic properties that are essential for spintronic functionality. Our study unveils light-field coherent control of spin dynamics and macroscopic magnetic moments in the initial non-dissipative temporal regime and establishes optical frequencies as the speed limit of future coherent spintronic applications, spin transistors and data storage media.

Conformational Equilibration Time of Unfolded Protein Chains and the Folding Speed Limit

The speed with which the conformers of unfolded protein chains interconvert is a fundamental question in the study of protein folding. Kinetic evidence is presented here for the time constant for interconversion of disparate unfolded chain conformations of a small globular protein, cytochrome c, in the presence of guanidine hydrochloride denaturant. The axial binding reactions of histidine and methionine residues with the Fe(II) heme cofactor were monitored with time-resolved magnetic circular dichroism spectroscopy after photodissociation of the CO complexes of unfolded protein obtained from horse and tuna and from several histidine mutants of the horse protein. A kinetic model fitting both the reaction rate constants and spectra of the intermediates was used to obtain a quantitative estimate of the conformational diffusion time. The latter parameter was approximated as a first-order time constant for exchange between conformational subensembles presenting either a methionine or a histidine residue to the heme iron for facile binding. The mean diffusional time constant of the wild type and variants was 3 +/- 2 mus, close to the folding "speed limit". The implications of the relatively rapid conformational equilibration time observed are discussed in terms of the energy landscape and classical pathway time regimes of folding, for which the conformational diffusion time can be considered a pivot point.

Earliest Events in Protein Folding: Submicrosecond Secondary Structure Formation in Reduced Cytochrome c,

Protein folding is uniquely characterized as a dynamic process by the tremendous conformational heterogeneity of the unfolded state. This conformational heterogeneity implies the possibility of significant kinetic heterogeneity in the folding dynamics, yet many folding reactions appear to proceed homogeneously, i.e., from unfolded conformations that are in equilibrium with each other and a transition state. At what point in time on the way to the folded state the kinetic heterogeneity of the unfolded state is lost to conformational equilibration has been an open question. We present evidence in this regard obtained from nanosecond far-UV optical rotatory dispersion spectroscopy of the fastest folding process observed in photoreduced cytochrome c, a secondary structure formation process proceeding on a submicrosecond time scale at high denaturant concentration (observed as a “burst” phase in millisecond stopped-flow circular dichroism studies). The kinetics of this fast folding process imply that it proceeds from a conformational ensemble that is not in equilibrium with the bulk of protein conformers during the time required to completely reduce the sample, ∼100 μs. We thus determine a lower limit on the time required for conformational equilibration of ∼10-4 s, in agreement with the previous estimate from time-resolved magnetic circular dichroism measurements on photolyzed cytochrome c-CO (Goldbeck, R. A.; Thomas, Y. G.; Chen, E.; Esquerra, R. M.; Kliger, D. S. Proc. Natl. Acad. Sci. 1999, 96, 2782−2787). Combined with an upper limit from stopped-flow measurements (Lyubovitsky, J. G.; Gray, H. B.; Winkler, J. R. J. Am. Chem. Soc. 2002, 124, 5481−5485), this allows us to bracket the conformational diffusion time, i.e., the time interval over which the kinetic heterogeneity of this protein's unfolded state is lost through conformational equilibration, within the range ∼10-4−10-3 s. This estimate of the conformational diffusion time implies that the earliest folding events, helix formation and possibly the collapse of extended conformations, proceed under an energy landscape regime wherein conformational diffusion is slow, whereas the final (milliseconds) folding phase proceeds along a classical kinetic pathway.

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.

Optical Analysis of an Ellipsometric Technique for Time-Resolved Magnetic Circular Dichroism Spectroscopy

The Mueller matrix calculus is used to derive signal equations for a technique for nanosecond time-resolved magnetic circular dichroism (TRMCD) spectroscopy that is based on detection of the polarization state of highly eccentric, elliptically polarized light. These equations link the observed signal to the magnetic circular dichroism of the sample in the presence of measurement artifacts associated with imperfect optical devices and the birefringence induced in laser-photolyzed samples by photoselection. The results provide a methodical basis for optimization of optics and artifact reduction. An experimental example of the most important artifact, arising from the second-order coupling of birefringence and Faraday rotation, is presented for the case of laser-photolyzed (carbonmonoxy)myoglobin

Time-resolved magnetic circular dichroism spectroscopy of photolyzed carbonmonoxy cytochrome c oxidase (cytochrome aa3)

Nanosecond time-resolved magnetic circular dichroism (TRMCD) and time-resolved natural circular dichroism (TRCD) measurements of photolysis products of the CO complex of eukaryotic cytochrome c oxidase (CcO-CO) are presented. TRMCD spectra obtained at 100 ns and 10 microseconds after photolysis are diagnostic of pentacoordinate cytochrome a3Fe2+, as would be expected for simple photodissociation. Other time-resolved spectroscopies (UV-visible and resonance Raman), however, show evidence for unusual Fea3(2+) coordination after CO photolysis (Woodruff, W. H., O. Einarsdóttir, R. B. Dyer, K. A. Bagley, G. Palmer, S. J. Atherton, R. A. Goldbeck, T. D. Dawes, and D. S. Kliger. 1991. Proc. Nat. Acad. Sci. U.S.A. 88:2588-2592). Furthermore, time-resolved IR experiments have shown that photodissociated CO binds to CuB+ prior to recombining with Fea3(2+) (Dyer, R. B., O. Einarsdóttir, P. M. Killough, J. J. López-Garriga, and W. H. Woodruff. 1989. J. Am. Chem. Soc. 111:7657-7659). A model of the CcO-CO photolysis cycle which is consistent with all of the spectroscopic results is presented. A novel feature of this model is the coordination of a ligand endogenous to the protein to the Fe axial site vacated by the photolyzed CO and the simultaneous breaking of the Fe-imidazole(histidine) bond.

Nature and functional implications of the cytochrome a3 transients after photodissociation of CO-cytochrome oxidase.

Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (approximately 1 microseconds) as the post-photodissociation CU+B-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CuB to Fea3 when CO binds to CuB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The time-resolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CuB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fea3 pair. We speculate as to the identity of the ligand that is transferred between CuB and Fea3 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.