Fourier Transform Infrared Characterization of the Azido Complex of Methane Monooxygenase Hydroxylase from Methylococcus capsulatus (Bath)

Abstract

The azido complex formed in oxidized methane monooxygenase from Methylococcus capsulatus (Bath) was investigated with resonance Raman and FTIR techniques. These experiments show the presence of a nuas(NNN) at approximately 2077 cm-1 which splits to two components at 2059 and 2073 cm-1 with 15N14N2. The vibrational data are assigned to an azido complex bound terminally to one iron(III) at the diiron center. When the azido complex is illuminated at 15 K, a new nuas(NNN) is observed at 2136 cm-1 which is assigned to a photodissociated HN3 within the substrate pocket. We propose a model where an aqua ligand engages a hydrogen bond interaction with the 1N atom of the azido group and acts as at a proton donor during the photolysis process.

The Production of Nitrous Oxide by the Heme/Nonheme Diiron Center of Engineered Myoglobins (FeBMbs) Proceeds through a trans-Iron-Nitrosyl Dimer

“…Low-temperature FTIR photolysis was conducted using a previously described method. − FTIR sandwich films were prepared as described above by loading approximately 15 μL of a 1 mM protein solution into an FTIR cell with a 15-μm Teflon spacer. After the formation of the NO adduct was confirmed by UV–vis absorption spectroscopy, the FTIR cell was mounted to a sample rod which was then flash-frozen in liquid N 2 and inserted into the sample compartment of a closed-cycle cryogenic system (Omniplex, Advanced Research Systems).…”

A Sensitive Multispectroscopic Probe for Nucleic Acids

“…Other groups have used one-and two-dimensional IR (2D IR) of the azide anion to probe protein dynamics. 9,10 Azide has also been used as an IR probe of methane monooxygenase 11 and photosystem II. 12 Nitrile probes 8 represent smaller perturbations to a system and less conformational flexibility compared to azide probes; however, the greater sensitivity to environment and larger extinction coefficient of the azido group suggests that azide probes can rival the utility of nitrile probes.…”

“…Polar to less polar transitions were observed for N 3 -F at position 250 (N 3 -F250), the reverse transition was found for N 3 −F227, and the environment around N 3 −F102 was essentially unchanged in going from dark to photoproduct, as expected. Other groups have used one- and two-dimensional IR (2D IR) of the azide anion to probe protein dynamics. , Azide has also been used as an IR probe of methane monooxygenase and photosystem II …”

Bioinorganic chemistry

“…75 The results of Fe K-edge and EXAFS analysis reveal subtle differences between the hydroxylases that may be correlated to access of the active site. The azido complex of MMOH from Methylococcus capsulatus (Bath) has been characterised by FTIR spectroscopy 76 and a rapid freeze-quench method in conjunction with Mo¨ssbauer spectroscopy has been used to monitor changes at the active site of methane monooxygenase during catalytic turnover. 77 The reaction between an Fe III complex and O 2 to afford an unprecedented, stable catalytically active diiron(IV)-m-oxo compound has been described as an analogue of the active site of MMOH, Fig.…”