The long-standing relationship between paramagnetic NMR and iron–sulfur proteins: the mitoNEET example. An old method for new stories or the other way around?

• Main Authors: F. Camponeschi, A. Gallo, M. Piccioli, L. Banci
• Format: Article
• Language: English
• Published: Copernicus Publications 2021-04-01
• Series: Magnetic Resonance
• Online Access: https://mr.copernicus.org/articles/2/203/2021/mr-2-203-2021.pdf

<p>Paramagnetic NMR spectroscopy and iron–sulfur (Fe–S) proteins have maintained a synergic relationship for decades. Indeed, the hyperfine shifts with their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues have been extensively used as a fingerprint of the type and of the oxidation state of the Fe–S cluster within the protein frame. The identification of NMR signals from residues surrounding the metal cofactor is crucial for understanding the structure–function relationship in Fe–S proteins, but it is generally impaired in standard NMR experiments by paramagnetic relaxation enhancement due to the presence of the paramagnetic cluster(s). On the other hand, the availability of systems of different sizes and stabilities has, over the years, stimulated NMR spectroscopists to exploit iron–sulfur proteins as paradigmatic cases to develop experiments, models, and protocols. Here, the cluster-binding properties of human mitoNEET have been investigated by 1D and 2D <span class="inline-formula">¹</span>H diamagnetic and paramagnetic NMR, in its oxidized and reduced states. The NMR spectra of both oxidation states of mitoNEET appeared to be significantly different from those reported for previously investigated <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M2" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><mo>[</mo><msub><mi mathvariant="normal">Fe</mi><mn mathvariant="normal">2</mn></msub><msub><mi mathvariant="normal">S</mi><mn mathvariant="normal">2</mn></msub><msup><mo>]</mo><mrow><mn mathvariant="normal">2</mn><mo>+</mo><mo>/</mo><mo>+</mo></mrow></msup></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="58pt" height="17pt" class="svg-formula" dspmath="mathimg" md5hash="79572f08ecb9017531a5864e5ff2fda1"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="mr-2-203-2021-ie00001.svg" width="58pt" height="17pt" src="mr-2-203-2021-ie00001.png"/></svg:svg></span></span> proteins. The protocol we have developed in this work conjugates spectroscopic information arising from “classical” paramagnetic NMR with an extended mapping of the signals of residues around the cluster which can be taken, even before the sequence-specific assignment is accomplished, as a fingerprint of the protein region constituting the functional site of the protein. We show how the combined use of 1D NOE experiments, <span class="inline-formula">¹³C</span> direct-detected experiments, and double- and triple-resonance experiments tailored using R<span class="inline-formula">₁</span>- and/or R<span class="inline-formula">₂</span>-based filters significantly reduces the “blind” sphere of the protein around the paramagnetic cluster. This approach provided a detailed description of the unique electronic properties of mitoNEET, which are responsible for its biological function. Indeed, the NMR properties suggested that the specific electronic structure of the cluster possibly drives the functional properties of different <span class="inline-formula">[Fe₂S₂]</span> proteins.</p>