Nuclear inelastic scattering of heme proteins: from iron ligand vibrations to low energy protein modes

Abstract

The binding of the signal molecule nitric oxide (NO) to the NO transporter protein Nitrophorin 2 (NP2) from the bloodsucking insect Rhodnius prolixus has been characterized by Mössbauer spectroscopy as well as nuclear forward scattering (NFS) and nuclear inelastic scattering (NIS). A striking feature of the vibrational spectrum obtained from NP2-NO is a vibration at 594 cm −1 . This mode is assigned to a Fe-NO stretching mode via simulation of the NIS data by density functional theory (DFT) coupled with molecular mechanics (MM) methods. At frequencies below 100 cm −1 collective motions like heme doming occur which could explain spectroscopic features observed by NIS at these low energies.

Electron Spin Density on the Axial His Ligand of High-Spin and Low-Spin Nitrophorin 2 Probed by Heteronuclear NMR Spectroscopy

“…NP2 in the absence of added ligand is bound to a water molecule at pH 6.5 and below, − and has been shown to be high-spin, S = 5/2, on the basis of EPR ( g y = 5.94, g x = 5.67, and g z = 1.99) and Mössbauer spectroscopy (δ = 0.30 ± 0.01 mms –1 , Δ E Q = 2.53 ± 0.01 mms –1 ) at 4.2 K. However, the 1 H NMR spectrum of ligand-free NP2 at 305 K, Figure , top, shows that the proton chemical shifts of the heme methyls of the aqua complex of NP2 at ambient temperatures are smaller than those of the aqua complexes of metMb, , horseradish peroxidase (HRP), , other peroxidases, inhibitor-bound heme oxygenase, , and bacterial ferricytochromes c ′. , The smaller chemical shifts of NP2 may be attributed to an effect of heme ruffling, which is known to be very large in nitrophorins. Another possibility is the existence of a high-spin ⇌ low-spin equilibrium, as the one reported for HasA. − The possibility of this phenomenon is currently under investigation and will be addressed in a future manuscript (R.E.B.…”

Dimerization of Nitrophorin 4 at Low pH and Comparison to the K1A Mutant of Nitrophorin 1

“…The sequences are shown in Figure S1 of the Supporting Information. These proteins have been investigated by a number of techniques (DOI: 10.1021/bi501305a), ,,− and the solid state structures of one or more ligand complexes of NP1, , NP2, , and NP4 − have been determined by X-ray crystallography. The structures are unique for heme proteins, in that the heme is located inside, but at the open end, of a β-barrel, , as shown in Figure , rather than in the more commonly observed largely α-helical globin or four-helix bundle folds.…”