DEER Sensitivity between Iron Centers and Nitroxides in Heme-Containing Proteins Improves Dramatically Using Broadband, High-Field EPR

Abstract

This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron–electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.

Pulsed EPR Dipolar Spectroscopy under the Breakdown of the High‐Field Approximation: The High‐Spin Iron(III) Case

“…The latter case has severala dvantages and applications: 1) the use of metal centers forP DS allows to reduce the number of spin labels and, consequently,t he number of protein mutations, 2) it enables orthogonal spin labeling [8] as the metal center has different spectroscopic properties than the majority of spin labels, 3) the distance constraintso btained from such PDS measurementse nable the localization of metal ions within the protein fold by trilateration [9,10] and the docking of different parts of protein complexes using metal ions as anchor points. [11] To date, PDS measurements have been applied to av ariety of different intrinsic metal centers, such as Cu 2 + , [12][13][14][15][16][17][18][19][20] Mn 2 + , [21][22][23][24][25] Co 2 + , [26,27] and low-spin Fe 3 + , [6,[28][29][30][31] as well as ironsulfur [17,[32][33][34] and manganese [35,36] clusters. In all these cases, the protocols for PSD measurements are well-established and the extractiono fd istance constraints from the corresponding PDS data can be readily achieved under the high-field approximation.…”

Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

“…Human neuroglobin is a good model system in this respect because both a high resolution X-ray structure [40] and DEER data [41,42] are available. Human neuroglobin is a good model system in this respect because both a high resolution X-ray structure [40] and DEER data [41,42] are available.…”

Pulsed Dipolar EPR Spectroscopy and Metal Ions: Methodology and Biological Applications

“…[83] Later, the SNR was improved by a factor of 30 using composite pulses at W-band. [84] However, even though a reasonable SNR could be achieved, an accurate conversion of the PELDOR time traces into a distance distributions may be obstructed by orientation selectivity effects. As was mentioned above, a common way to account for the orientation selectivity is to measure several PELDOR time traces for different orientations of an anisotropic spin and then to analyze all time traces together.…”