P +

Mo nitrogenase consists of two component proteins: the Fe protein, which contains a [Fe(4)S(4)] cluster, and the MoFe protein, which contains two different classes of metal cluster: P-cluster ([Fe(8)S(7)]) and FeMoco ([Mo-Fe(7)S(9)C•Homocitrate]). The P-cluster is believed to mediate the electron transfer between the Fe protein and the MoFe protein via inter-conversions between its various oxidation states, such as the all-ferrous state (P(N)) and the one (P(+))- and two (P(2+))-electron oxidized states. While the structural and electronic properties of P(N) and P(2+) states have been well characterized, little is known about the electronic structure of the P(+) state. Here, a mutant strain of Azotobacter vinelandii (DJ1193) was used to facilitate the characterization of the P(+) state of P-cluster. This strain expresses a MoFe protein variant (designated ΔnifB β-188(Cys) MoFe protein) that accumulates the P(+) form of P-cluster in the resting state. MCD spectrum of the P-cluster in the oxidized ΔnifB β-188(Cys) MoFe protein closely resembles that of the P(2+) state in the oxidized wild-type MoFe protein, except for the absence of a major charge-transfer band centered at 823 nm. Moreover, magnetization curves of ΔnifB β-188(Cys) and wild-type MoFe proteins suggest that the P(2+) species in both proteins have the same spin state. MCD spectrum of the P(+) state in the ΔnifB β-188(Cys) MoFe protein, on the other hand, is associated with a classic [Fe(4)S(4)](+) cluster, suggesting that the P-cluster could be viewed as two coupled 4Fe clusters and that it could donate either one or two electrons to FeMoco by using one or both of its 4Fe halves. Such a mode of action of P-cluster could provide energetic and kinetic advantages to nitrogenase in the complex mechanism of N(2) reduction

Integrated approaches to unravel the impact of protein lipoxidation on macromolecular interactions

15 p.-5 fig.-3 tab.Protein modification by lipid derived reactive species or lipoxidation is increased during oxidative stress, a common feature observed in many pathological conditions. Biochemical and functional consequences of lipoxidation include changes in the conformation and assembly of the target proteins, altered recognition of ligands and/or cofactors, changes in the interactions with DNA or protein-protein interactions, modifications in membrane partitioning and binding and/or subcellular localization. These changes may impact, directly or indirectly, signaling pathways involved in the activation of cell defense mechanisms, but when these are overwhelmed they may lead to pathological outcomes. Mass spectrometry provides state of the art approaches for the identification and characterization of lipoxidized proteins/residues and the modifying species. Nevertheless, understanding the complexity of the functional effects of protein lipoxidation requires the use of additional methodologies. Herein, biochemical and biophysical methods used to detect and measure functional effects of protein lipoxidation at different levels of complexity, from in vitro and reconstituted cell-like systems to cells, are reviewed, focusing especially on macromolecular interactions. Knowledge generated through innovative and complementary technologies will contribute to comprehend the role of lipoxidation in pathophysiology and, ultimately, its potential as target for therapeutic intervention.This work was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement number 675132 (http://cordis.europa.eu/project/rcn/198275_en.html), and by grants from the Spanish Ministerio de Economía y Competitividad (MINECO/FEDER, http://www.mineco.gob.es/portal/site/mineco/idi) SAF2015-68590-R to DPS and BFU2016-75471-C2-1-P to GR, and RETIC Aradyal from ISCIII/FEDER (RD16/0006/0021) to DPS.Peer reviewe