A Multifrequency HYSCORE Study of Weakly Coupled Nuclei in Frozen Solutions of High-Spin Aquometmyoglobin

Abstract

In this work, we show the extreme power of multifrequency HYSCORE (hyperfine sublevel correlation spectroscopy) techniques to unravel the hyperfine interactions of the electron spin with the remote nuclei in the heme site of high-spin ferric heme proteins. Horse heart aquo-metmyoglobin was used as a model system to demonstrate the power of these techniques. Experimental evidence was collected and assigned to protons of the proximal histidine ligand, to the mesoprotons of the heme ligand, and to two different protons of the distal water ligand. The latter difference relates to the stabilization of the water ligand by the E7His residue. Furthermore, HYSCORE signals of the remote N(delta) of the proximal (F8) and N(epsilon) of the distal (E7) histidine were detected. Finally, correlation peaks from the lesser-abundant (13)C nuclei of the heme ligand could be detected. These novel results allow dissection of the hyperfine couplings into individual contributions and calculation of the spin density in the pi and sigma orbitals, thus completing earlier electron paramagnetic resonance and liquid-state NMR data.

In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase—The Interplay of Key Amino Acids, pH, Buffer and Glycerol

“…Glycerol was needed in this case as glassing agent to increase the electronic relaxation times and allow HYSCORE experiments. The overall HYSCORE features of the different Kp DyP variants are qualitatively in agreement with what was observed for other high-spin heme systems [ 37 , 38 , 39 , 40 , 41 , 42 ]. While the HYSCORE results are essentially the same for WT and D143A Kp DyP, subtle but significant difference were observed for R232A.…”

“…A previous in-depth study of aquometmyoglobin showed that the cross peaks related to the interaction of the electron spin with the N ε of the proximal histidine are usually weak or suppressed in the standard HYSCORE spectra, as is also observed here. At other magnetic-field settings, the matched 1 H HYSCORE spectrum reveals the characteristic cross-peaks due to the protons at the C ε and C δ positions of the proximal His ( Figure S8 ) [ 40 ].…”

Structure determination of bound nitrogen-based adducts with copper(ii) acetylacetonato; an EPR, ENDOR and DFT study

“…Unfortunately, we were unable to resolve any 14 N ENDOR resonances. In principle, the small couplings expected from the weakly interacting nitrogen nucleus in either coordination mode (i.e., A( 14 N py ) = [À1.14, À1.14, À0.53] MHz, Q( 14 N py ) = [0.44, 1.16, À1.60] MHz and A( 14 N NH 2 ) = [À0.82, À0.82, 0.26] MHz, Q( 14 N NH 2 ) = [0.82, 1.24, À2.06] MHz as predicted from the DFT calculations) should be resolvable by HYSCORE measurements, 25,41 although in this contribution we have focussed on the information available from the 1 H ENDOR data.…”

A sensitivity leap for X-band EPR using a probehead with a cryogenic preamplifier

“…In addition, overlapping HYSCORE signals require spectral subtraction [26] or relaxation filters [21,27]. More sensitive hyperfine measurements at Qband cannot substitute those at X-band as frequently both bands complement each other [24,25,28,29]. The SMART-HYSCORE sequence has been proposed to avoid blind spots and enable correlation of frequencies stemming from different nuclei [21,30], however, it usually necessitates long acquisition times on proteins (e.g.…”