Author

Shen Lu

Date

October 2005

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Environmental and Biomolecular Systems

Institution

Oregon Health & Science University

Abstract

Our research goal is to understand how diiron metal clusters control the diversity of their reaction with 0[subscript]2 and NO. X-ray crystal structures of protein R2 from E. coli ribonucleotide reductase and the hydroxylase component of methane monooxygenase (MMOH) from Methyloccocus capsulatus (Bath) have revealed very similar non-heme diiron carboxylated bridged cluster. Yet, these two enzymes react very differently with 0[subscript]2. In single turnover reaction, a diiron(III) peroxo intermediate is spectroscopically observable in MMOH but not in wt-R2, suggesting that the divergence between the 0[subscript]2-reaction of MMOH and R2 might occur at the onset of the reaction. To investigate the possible involvement of a peroxo intermediate species in the 0[subscript]2 activation of wt-R2, and determine the geometry of 0[subscript]2-binding in R2 and MMOH, we have characterized 0[subscript]2-analogs in these proteins using UV-vis absorption, EPR, RR, and FTIR spectroscopies. Our investigation of the reaction of NO with the diiron sites in wt-R2 and the variant proteins D84E-R2, and W48F/D84E-R2 shows that in these three proteins the diiron sites accommodate two NO molecules to form a spin coupled {FeNO}[superscript]7 dimer. Both Fe-N-0 units share a common vibrational signature indicative of a fully symmetric [{FeNo}[superscript]7][subscript]2 cluster. These structural motifs are consistent with the symmetric bridging, peroxo intermediates characterized in the variant proteins, and they suggest that while such a symmetric peroxo intermediate is not detectable in wt- R2, the binding of O[subscript]2 and its initial reductive activation is the same in all three proteins. The binding of azide to oxidized MMOH was also investigated with RR and FTIR techniques. The FTIR spectra define the azido complex as terminally bound to only one iron (III) at the diiron center. H/D exchange experiments detect the presence of a hydrogen bond interaction at the coordinating (N1) azide nitrogen atom. The hydrogen bond partner is proposed to be an aqua ligand on the second Fe. The photodissociation of the azido group from the metal cluster is associated with a proton uptake to from hydrazoic acid. The proton donor to the azido group is likely to be the same group involved in the hydrogen bond interaction. Our experimental data provide structural details that can be compared to theoretical prediction of the O[subscript]2 reaction in MMOH. The structure of the azide complex suggest that the peroxo complex might adopt an asymmetric structure, and that as predicted by the theoretical analysis, the presence of hydrogen bond between the peroxo group and an aqua ligand might favor the decay of H[subscript]peroxo and the formation of higher valent intermediate as the 0-0 bond is cleaved. The reaction of NO with the diiron cluster of two NO reductases, cNOR from Paracoccus denitrijcans and [subscript]qCu[subscript]ANOR from Bacillus azotoformans, were also studied. Here again, the 2-electron reduction of two NO molecules to from N[subscript]20 is poorly understood. To determine how NO initially binds at the active site, we studied the binding of CO as an analog to NO-binding. These experiments reveal that two CO molecules could bind concomitantly at the diiron (II) sites, to form a heme-CO complex and a non-heme Fe[subscript]BCO complex. Our FTIR experiments strongly supported a mechanistic model where two NO molecules form a [{FeNO}[superscript]7][subscript]2 unit that promote N-N bond formation.

Identifier

doi:10.6083/M4N29TWD

School

OGI School of Science and Engineering

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