No abstract

Nitric oxide (NO) is an important signaling molecule that is involved in a wide range of physiological and pathological events in biology. Metal coordination chemistry, especially with iron, is at the heart of many biological transformations involving NO. A series of heme proteins, nitric oxide synthases (NOS), soluble guanylate cyclase (sGC), and nitrophorins, are responsible for the biosynthesis, sensing, and transport of NO. Alternatively, NO can be generated from nitrite by heme- and copper-containing nitrite reductases (NIRs). The NO-bearing small molecules such as nitrosothiols and dinitrosyl iron complexes (DNICs) can serve as an alternative vehicle for NO storage and transport. Once NO is formed, the rich reaction chemistry of NO leads to a wide variety of biological activities including reduction of NO by heme or non-heme iron-containing NO reductases and protein post-translational modifications by DNICs. Much of our understanding of the reactivity of metal sites in biology with NO and the mechanisms of these transformations has come from the elucidation of the geometric and electronic structures and chemical reactivity of synthetic model systems, in synergy with biochemical and biophysical studies on the relevant proteins themselves. This review focuses on recent advancements from studies on proteins and model complexes that not only have improved our understanding of the biological roles of NO but also have provided foundations for biomedical research and for bio-inspired catalyst design in energy science.

Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of the Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres and nuclearity, all cupredoxins share a common Greek Key β-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, α3DChC2.1 While this design demonstrated that a β-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide α3D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRANDα3D (GRα3D) with ΔGu = −11.4 kcal/mol compared to the original design’s −5.1 kcal/mol. Diffraction quality crystals were grown of GRα3D (a first for an α3D peptide) and solved to a resolution of 1.34 Å. Examination of this structure suggested that Glu41 might interact with the Cu in our previously reported red copper protein. The previous bis(histidine)(cysteine) site (GRα3DChC2) was designed into this new scaffold and a series of variant constructs were made to explore this hypothesis. Mutation studies around Glu41 not only prove the proposed interaction, but also enabled tuning of the constructs’ hyperfine coupling constant from 160 to 127 × 10−4 cm−1. X-ray absorption spectroscopy analysis is consistent with these hyperfine coupling differences being the result of variant 4p mixing related to coordination geometry changes. These studies not only prove that an Glu41–Cu interaction leads to the α3DChC2 construct’s red copper protein like spectral properties, but also exemplify the exact control one can have in a de novo construct to tune the properties of an electron-transfer Cu site.

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Rev-Erbβ is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation. Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind other proteins, and its activity in gas sensing. Rev-Erbβ binds Fe3+-heme more tightly than Fe2+-heme, suggesting its activities may be regulated by the heme redox state. Yet, this critical role of heme redox chemistry in defining the protein’s resting state and function is unknown. We demonstrate by electrochemical and whole-cell electron paramagnetic resonance experiments that Rev-Erbβ exists in the Fe3+ form within the cell allowing the protein to be heme replete even at low concentrations of labile heme in the nucleus. However, being in the Fe3+ redox state contradicts Rev-Erb’s known function as a gas sensor, which dogma asserts must be Fe2+. This paper explains why the resting Fe3+ state is congruent both with heme binding and cellular gas sensing. We show that the binding of CO/NO elicits a striking increase in the redox potential of the Fe3+/Fe2+ couple, characteristic of an EC mechanism in which the unfavorable Electrochemical reduction of heme is coupled to the highly favorable Chemical reaction of gas binding, making the reduction spontaneous. Thus, Fe3+-Rev-Erbβ remains heme-loaded, crucial for its repressor activity, and undergoes reduction when diatomic gases are present. This work has broad implications for proteins in which ligand-triggered redox changes cause conformational changes influencing its function or interprotein interactions (e.g., between NCoR1 and Rev-Erbβ). This study opens up the possibility of CO/NO-mediated regulation of the circadian rhythm through redox changes in Rev-Erbβ.

Manganese porphyrins are used as catalysts in the oxidation of olefins and nonactivated hydrocarbons. Key to these reactions are high-valent Mn−(di)oxo species, for which [Mn(Porph)(X)] serve as precursors. To elucidate their properties, it is crucial to understand the interaction of the Mn center with the porphyrin ligand. Our study focuses on simple high-spin [Mn III (TPP)X] (X = F, Cl, I, Br) complexes with emphasis on the spectroscopic properties of [Mn III (TPP)Cl], using variabletemperature variable-field magnetic circular dichroism spectroscopy and time-dependent density functional theory to help with band assignments. The optical properties of [Mn III (TPP)Cl] are complicated and unusual, with a Soret band showing a high-intensity feature at 21050 cm −1 and a broad band that spans 23200−31700 cm −1 . The 15000−18500 cm −1 region shows the Cl(p x/y ) → d π (CT (Cl,π) ), Q band, and overlap-forbidden Cl(p x/y )_d π → d x 2 −y 2 transitions that gain intensity from the strongly allowed π → π* (0) transition. The 20000−21000 cm −1 region displays the prominent pseudo A-type signal of the Soret band. The strongly absorbing features at 22500−28000 cm −1 exhibit A 1u ⟨79⟩/A 2u ⟨81⟩ → d π , CT (Cl,π/σ) , and symmetry-forbidden CT character, mixed with the π → π* (0) transition. The strong d x 2 −y 2 _B 1g ⟨80⟩ orbital interaction drives the ground-state MO mixing. Importantly, the splitting of the Soret band is explained by strong mixing of the porphyrin A 2u (π)⟨81⟩ and the Cl(p z )_d z 2 orbitals. Through this direct orbital pathway, the π → π* (0) transition acquires intrinsic metal-d → porphyrin CT character, where the π → π* (0) intensity is then transferred into the high-energy CT region of the optical spectrum. The heavier halide complexes support this conclusion and show enhanced orbital mixing and drastically increased Soret band splittings, where the 21050 cm −1 band shifts to lower energy and the high-energy features in the 23200−31700 cm −1 range increase further in intensity, compared to the chloro complex.

Coupled dinuclear copper oxygen cores (Cu 2 O 2 ) featured in type III copper proteins (hemocyanin, tyrosinase, catechol oxidase) are vital for O 2 transport and substrate oxidation in many organisms. m-1,2-cis peroxidod icopper cores ( C P)h ave been proposed as key structures in the early stages of O 2 binding in these proteins;their reversible isomerization to other Cu 2 O 2 cores are directly relevant to enzyme function. Despite the relevance of such species to type III copper proteins and the broader interest in the properties and reactivity of bimetallic C P cores in biological and synthetic systems,t he properties and reactivity of C P Cu 2 O 2 species remain largely unexplored. Herein, we report the reversible interconversion of m-1,2-trans peroxido ( T P)a nd C P dicopper cores.C a II mediates this process by reversible binding at the Cu 2 O 2 core,h ighlighting the unique capability for metal-ion binding events to stabilizenovel reactive fragments and control O 2 activation in biomimetic systems.

9Rev-Erbβ is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation. [1][2][3][4][5][6][7] 10 Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind 11 other proteins, and its activity in gas sensing. [8][9][10][11] Rev-Erbβ binds Fe 3+ -heme tighter than Fe 2+ -12 heme, suggesting its activities may be regulated by the heme redox state. 9 Yet, this critical role of 13 heme redox chemistry in defining the protein's resting state and function is unknown. We 14 demonstrate by electrochemical and whole-cell electron paramagnetic resonance experiments that 15 Rev-Erbβ exists in the Fe 3+ form within the cell essentially allowing the protein to be heme-replete 16 even at low concentrations of labile heme in the nucleus. However, being in the Fe 3+ redox state 17 contradicts Rev-Erb's known function as a gas sensor, which dogma asserts must be a Fe 2+ protein 18 This paper explains why the resting Fe 3+ -state is congruent both with heme-binding and cellular 19 gas sensing. We show that the binding of CO/NO elicits a striking increase in the redox potential 20 of the Fe 3+ /Fe 2+ couple, characteristic of an EC mechanism in which the unfavorable 21 Electrochemical reduction of heme is coupled to the highly favorable Chemical reaction of gas 22 binding, making the reduction spontaneous. Thus, Fe 3+ -Rev-Erbβ remains heme-loaded, crucial 23 for its repressor activity, and only undergoes reduction when diatomic gases are present. This work 24 has broad implications for hemoproteins where ligand-triggered redox changes cause 25 conformational changes influencing protein's function or inter-protein interactions, like NCoR1 26 for Rev-Erbβ. This study opens up the possibility of CO/NO-mediated regulation of the circadian 27 rhythm through redox changes in Rev-Erbβ. 28 Introduction 29Rev-Erb-a and -β belong to the nuclear receptor superfamily. They are indispensable components 30 of the circadian rhythm and regulate the expression of genes involved in lipid and glucose 31 metabolism and inflammatory responses. [1][2][3][4][5][6][7]11,12 Rev-Erbs are also implicated in influencing 32 cognitive and neuronal functions. [13][14][15] Nuclear receptors have a characteristic N-terminal AB 33 region followed by a DNA binding C-domain linked to a ligand binding domain (LBD) via a 34 flexible linker. 16 Heme binding to the LBD promotes proteasomal protein degradation, facilitates 35 its interaction with co-repressors, and enhances its transcriptional repression activity. 8,9,11,17 36 In Rev-Erbβ-LBD, a Cys-Pro heme regulatory motif affords the Cys as an axial ligand to Fe 3+ -37 heme while a second axial ligation is provided by a distal Histidine (His) residue ( Fig. 1a). 18,19 38 Besides adopting a His/Cys ligated 6-coordinate low-spin (LS) Fe 3+ state, Rev-Erbβ can undergo 39 a thiol-disulfide redox switch wherein the coordinating Cys384 forms a disulfide bond with 40 Cys374 giving rise to a His/neutral residue ligated Fe 3+ heme state. 18,20 A me...