Exploring second coordination sphere effects in nitric oxide synthase

Abstract

Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the ‘Cys pocket’. These hydrogen bonding networks are generally believed to regulate the Fe–S interaction. Previous work identified the S(Cys) → Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) → Fe σ CT band in the LT MCD spectrum at about 27,800 cm−1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe–S(Cys) bond strength, at least in the hs ferric form of the protein studied here.

Electron Paramagnetic Resonance Spectroscopy as a Probe of Hydrogen Bonding in Heme-Thiolate Proteins

“…We hypothesize that a key contributing factor to functional divergence in heme-thiolate proteins is the structure of the hydrogen-bonding (H-bonding) environment surrounding the coordinating Cys­(thiolate) (Cys­(S – )) ligand. A well-defined H-bonding pocket envelops the heme-bound Cys­(S) in cytochrome P450 enzymes (a large group of type-1 heme-thiolate proteins), and a growing body of work implicates this H-bonding network in maintaining stability of the Fe–S bond and in modulating reactivity of the heme. − In a number of type-2 heme-thiolate proteins, H-bonding interactions may facilitate ligand switching at the heme, − although structural characterization of these interactions is limited.…”

Artificial Metalloproteins for Binding and Stabilization of a Semiquinone Radical

“… 1 − 12 In addition to their potential for application in synthesis, they are also valuable tools for the investigation of the role of the second coordination sphere in enzyme catalysis. 3 , 13 − 18 Furthermore, designed metalloproteins may allow for the stabilization of highly reactive compounds, giving rise to increased lifetimes of these species. Some metal semiquinone intermediates have been identified in natural enzymes and synthetic mimic complexes.…”

d‐Orbital Reconstructions Forced by Double Bow‐Shaped Deformations and Second Coordination Sphere Effects of Cu(II) Heme Analogs in HER**

“…This phenomenon indicates the double bow‐shaped architecture is able to stabilize the axial ligand inside the pocket and this effect increases with the increase of porphyrin deformation. Second(ary) coordination sphere (SCS) effects in proteins and transition metal complexes which correspond to interactions of the metal center and its primary ligands with groups that are not part of the intimate coordination environment of the metal have drawn increasing attentions [39–43] . The most important types of interactions are hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π‐stacking of aromatic side chains which are used by proteins for substrate binding [43] .…”

“…Second(ary) coordination sphere (SCS) effects in proteins and transition metal complexes which Chemistry-A European Journal correspond to interactions of the metal center and its primary ligands with groups that are not part of the intimate coordination environment of the metal have drawn increasing attentions. [39][40][41][42][43] The most important types of interactions are hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic side chains which are used by proteins for substrate binding. [43] As a well-known example, the distal His in hemoglobin (Hb) and myoglobin (Mb) which forms hydrogen bonding with dioxygen to stabilize the oxy-Hb/ oxy-Mb complex has inspired the development of picket fence porphyrins [44] where the four pivalamido groups afford strong trapping effect to O 2 , for example the oxygenation of [M-(TpivPP)(RIm)] (M=Co or Fe; RM = 1-MeIm, 2-MeHIm) porphyrins.…”

“…[39][40][41][42][43] The most important types of interactions are hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic side chains which are used by proteins for substrate binding. [43] As a well-known example, the distal His in hemoglobin (Hb) and myoglobin (Mb) which forms hydrogen bonding with dioxygen to stabilize the oxy-Hb/ oxy-Mb complex has inspired the development of picket fence porphyrins [44] where the four pivalamido groups afford strong trapping effect to O 2 , for example the oxygenation of [M-(TpivPP)(RIm)] (M=Co or Fe; RM = 1-MeIm, 2-MeHIm) porphyrins. [45,46] Analogous to picket fence porphyrin, in the current study, 1-Cu presents a new example where the double bow strings and the pronounced saddle conformation of the porphyrin plane have built up a four-walled, half-ball pocket sphere to trap the attacking substrates (Figure 6b).…”