Spectroscopic evidence for conformational relaxation in myoglobin.

Kriegl

Journal of Biological Chemistry

2004

132

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4 -11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His 64 and Lys 67 . His 64 interferes with exogenous ligand access to the heme iron. Lys 67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.

Section: Resultsmentioning

confidence: 99%

Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding

Kriegl

Journal of Biological Chemistry

2004

132

Proc. Natl. Acad. Sci. U.S.A.

“…In Mb*CO, the iron has moved out of the heme plane by 80% (2). In the relaxation to deoxy Mb (Mb*CO 3 Mb), the barriers for recombination increase markedly (14,(25)(26)(27)(28), while the iron shifts only by another 0.05 Å away from the mean heme plane (2). To identify the coordinate that is correlated to the rebinding barrier, we should direct our attention to structural features in the vicinity of the heme that do not change in the first transition following photolysis (MbCO 3 Mb*CO), but do change significantly in the relaxation (Mb*CO 3 Mb).…”

Section: Discussionmentioning

confidence: 99%

Structural factors controlling ligand binding to myoglobin: A kinetic hole-burning study

Ormos

Száraz

Cupane

et al. 1998

Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CObound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model parameters that characterize the homogeneous subensembles within the conformationally heterogeneous ensemble. Such couplings between spectral and functional parameters arise when they both originate from a common structural coordinate. This effect is frequently denoted as ''kinetic hole burning.'' The study of these correlations gives direct insights into the structure-function relationship in proteins. On the basis of earlier work that assigned spectral parameters to geometric properties of the heme, the connections with the heme geometry are discussed. We show that two separate structural coordinates inf luence the Soret line shape, but only one of the two is coupled to the enthalpy barrier for rebinding. We give evidence that this coordinate, contrary to widespread belief, is not the iron displacement from the mean heme plane.

Proc. Natl. Acad. Sci. U.S.A.

“…More direct methods, such as x-ray scattering (7) and Mössbauer spectroscopy (for review, see ref. 1) and spectroscopic and kinetic hole burning (10)(11)(12), have indeed detected such populations of slightly differing conformations.…”

mentioning

confidence: 99%

Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates

Dioumaev

Lanyi

2007

The time course of thermal reactions after illumination of 100% humidified bacteriorhodopsin films was followed with FTIR spectroscopy between 125 and 195 K. We monitored the conversion of the initial photoproduct, K, to the next, L intermediate, and a shunt reaction of the L state directly back to the initial BR state. Both reactions can be described by either multiexponential kinetics, which would lead to apparent end-state mixtures that contain increasing amounts of the product, i.e., L or BR, with increasing temperature, or distributed kinetics. Conventional kinetic schemes that could account for the partial conversion require reversible reactions, branching, or parallel cycles. These possibilities were tested by producing K or L and monitoring their interconversion at a single temperature and by shifting the temperature upward or downward after an initial incubation and after their redistribution. The results are inconsistent with any conventional scheme. Instead, we attribute the partial conversions to the other alternative, distributed kinetics, observed previously in myoglobin, which arise from an ensemble of frozen conformational substates at the cryogenic temperatures. In this case, the time course of the reactions reflects the progressive depletion of distinct microscopic substates in the order of their increasing activation barriers, with a distribution width for K to L reaction of Ϸ7 kJ/mol. low-temperature kinetics ͉ distributed kinetics ͉ photointermediates ͉ infrared ͉ FTIR