Characterization of Heme Orientational Disorder in a Myoglobin Reconstituted with a Trifluoromethyl-Group-Substituted Heme Cofactor

Abstract

The orientation of a CF-substituted heme in sperm whale myoglobin and L29F, H64L, L29F/H64Q, and H64Q variant proteins has been investigated using F NMR spectroscopy to elucidate structural factors responsible for the thermodynamic stability of the heme orientational disorder, i.e., the presence of two heme orientations differing by a 180° rotation about the 5-15 meso axis, with respect to the protein moiety. Crystal structure of the met-aquo form of the wild-type myoglobin reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-trifluoromethylporphyrinatoiron(III), determined at resolution of 1.25 Å, revealed the presence of the heme orientational disorder. Alterations of the salt bridge between the heme 13-propionate and Arg45(CD3) side chains due to the mutations resulted in equilibrium constants of the heme orientational disorder ranging between 0.42 and 1.4. Thus, the heme orientational disorder is affected by the salt bridge associated with the heme 13-propionate side chain, confirming the importance of the salt bridge in the heme binding to the protein.

Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions

“…This requires binding of substrates at relatively unique conformations. However, it has been shown that ligand binding to proteins might involve some level of heterogeneity ( 28 ). Enzymes with several substrates, like AK, bind them while in an open conformation and form the proper configuration of the active site only after binding ( 29 ).…”

Detecting rotational disorder in heme proteins: A comparison between resonance Raman spectroscopy, nuclear magnetic resonance, and circular dichroism

“…[6,7,12,[50][51][52][53] Other studies on Mb reconstituted with artificially modified hemins supported the idea that the contacts of the peripheral substituents, especially the vinyl groups, with the residues of the heme pocket are much more important in determining the preferential heme orientation than the propionate-protein salt bridges. [54] More recently, the crucial role of steric interactions between the residue side chains and the heme peripheral groups in heme binding was identified, [55,56] favoring the intramolecular reorientation mechanism within the Mb heme crevice over the intermolecular mechanism. [56,57]…”

“…Usually, the amount of reversed heme detected is quite small and its presence depends not only on the specific globin, but also on the oxidation and ligation states of the heme iron. The nature of the ligands has also been shown to be important [ 1–3,6–8,13–20 ] and the reversed heme orientation can affect the activity or the functional properties of the proteins. [ 21–25 ] For example, it was reported that the two conformer populations of native oxidized myoglobin (Mb) unfold and refold following two parallel kinetic reactions with rate constants differing 10‐fold, despite having identical absorption spectra and equilibrium thermodynamic stability.…”

“…The heme rotational disorder has been identified through reconstitution studies of wild type and mutant proteins, with both native and a variety of chemically‐modified hemes. The studies have been carried out predominantly by the spectroscopic techniques of circular dichroism (CD), [ 9,18,19,38,39 ] NMR, [ 1,2,6–8,10–13,35 ] and more recently resonance (RR) spectroscopy, [ 21,40–43 ] these three techniques providing complementary information.…”

Neutron scattering study of the orientational disorder in potassium cyanide

“…6 Often, at least in the first phases found on cooling below the melting point, the disorder is dynamic, with the molecules or molecular ions undergoing rotational motion across a continuous distribution of molecular orientations, or jump rotations between a well-defined set of distinct orientations. Examples range from molecular crystals [1] such as fullerenes [2] to polyatomic ions in metal-organic frameworks [3] and even hemes in proteins [4]. Orientational disorder may lead to anomalous materials properties, such as reversible amorphisation under pressure [5] and giant barocaloric effects [6].…”