Spectroscopic Characterization of Mononitrosyl Complexes in Heme–Nonheme Diiron Centers within the Myoglobin Scaffold (Fe_BMbs): Relevance to Denitrifying NO Reductase

Abstract

Denitrifying NO reductases are evolutionarily related to the superfamily of heme-copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N2O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines, and Val-68 to a glutamic acid to create a nonheme FeB site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV-vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and CuI- or ZnII-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}7 species observable at liquid nitrogen temperature, the FeII-loaded proteins are EPR silent at ≥ 30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using 15NO and 15N18O. The apo- and CuI-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of FeII at the FeB site shifts the heme ν(FeNO) by +17 cm-1 and the ν(NO) by -50 cm-1 to 1549 cm-1. This low ν(NO) is without precedent for a six-coordinate heme {FeNO}7 species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme FeII. Detection of a similarly low ν(NO) in the ZnII-loaded protein supports this interpretation.

Steady-state kinetics with nitric oxide reductase (NOR): New considerations on substrate inhibition profile and catalytic mechanism

“…Besides inorganic model compounds [32] (and references therein), an engineered sperm whale myoglobin was constructed [33,34]. It successfully reproduced the NOR active centre with the magnetic coupling between the two iron sites, but experiments carried out in the presence of NO have shown low reductase activity [33][34][35]. Recently, Resonance Raman Spectroscopy was used to investigate the NO reduction mechanism in these modified myoglobins, and the formation of a stable heme-NO complex as a reaction intermediate was pointed.…”

Conversion of Nitric Oxide into Nitrous Oxide as Triggered by the Polarization of Coordinated NO by Hydrogen Bonding

“…[7] In bioengineered non-heme/heme NOR, the O NO atom of heme-NO electrostatically interacts with adistal metal ion, such as Zn 2+ or Fe 2+ ,inthe non-heme pocket, which also decreases the n NO value up to 50 cm À1 . [20] As DFT calculations on heme-copper oxidoreductase showed that the NO reduction proceeds through an NÀN coupling phase facilitated by the protonation of heme-NO to form an HN 2 O 2 À intermediate,w ep ostulated that treatment with CD 3 OD induced the formation of 2···CD 3 OD (shifted to n NO = 1513 cm À1 )t hrough Hbonding.I ti st his polarization that stimulates the interaction between 2 and 2···CD 3 OD and allows NÀNcoupling to proceed and form aplausible [N 2 O 2 ]bridged intermediate.…”

Effect of Outer-Sphere Side Chain Substitutions on the Fate of the trans Iron–Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (FeBMbs): Insights into the Mechanism of Denitrifying NO Reductases

“…(17) In addition, we found that equally low N-O stretching frequencies are observed when their Fe B sites are occupied with either Fe(II) or Zn(II), but not with Cu(I), suggesting that the 6cLS heme nitroxyl-like complexes are stabilized by electrostatic interactions with the divalent metal center at their Fe B sites. (17)…”