Copper-sulfenate complex from oxidation of a cavity mutant of Pseudomonas aeruginosa azurin

Metal-sulfenate centers are known to play important roles in biology and yet only limited examples are known due to their instability and high reactivity. Herein we report a copper-sulfenate complex characterized in a protein environment, formed at the active site of a cavity mutant of an electron transfer protein, type 1 blue copper azurin. Reaction of hydrogen peroxide with Cu(I)-M121G azurin resulted in a species with strong visible absorptions at 350 and 452 nm and a relatively low electron paramagnetic resonance gz value of 2.169 in comparison with other normal type 2 copper centers. The presence of a side-on copper-sulfenate species is supported by resonance Raman spectroscopy, electrospray mass spectrometry using isotopically enriched hydrogen peroxide, and density functional theory calculations correlated to the experimental data. In contrast, the reaction with Cu(II)-M121G or Zn(II)-M121G azurin under the same conditions did not result in Cys oxidation or copper-sulfenate formation. Structural and computational studies strongly suggest that the secondary coordination sphere noncovalent interactions are critical in stabilizing this highly reactive species, which can further react with oxygen to form a sulfinate and then a sulfonate species, as demonstrated by mass spectrometry. Engineering the electron transfer protein azurin into an active copper enzyme that forms a copper-sulfenate center and demonstrating the importance of noncovalent secondary sphere interactions in stabilizing it constitute important contributions toward the understanding of metal-sulfenate species in biological systems

Copper-sulfenate complex from oxidation of a cavity mutant of Pseudomonas aeruginosa azurin

Metal-sulfenate centers are known to play important roles in biology and yet only limited examples are known due to their instability and high reactivity. Herein we report a copper-sulfenate complex characterized in a protein environment, formed at the active site of a cavity mutant of an electron transfer protein, type 1 blue copper azurin. Reaction of hydrogen peroxide with Cu(I)-M121G azurin resulted in a species with strong visible absorptions at 350 and 452 nm and a relatively low electron paramagnetic resonance gz value of 2.169 in comparison with other normal type 2 copper centers. The presence of a side-on copper-sulfenate species is supported by resonance Raman spectroscopy, electrospray mass spectrometry using isotopically enriched hydrogen peroxide, and density functional theory calculations correlated to the experimental data. In contrast, the reaction with Cu(II)-M121G or Zn(II)-M121G azurin under the same conditions did not result in Cys oxidation or copper-sulfenate formation. Structural and computational studies strongly suggest that the secondary coordination sphere noncovalent interactions are critical in stabilizing this highly reactive species, which can further react with oxygen to form a sulfinate and then a sulfonate species, as demonstrated by mass spectrometry. Engineering the electron transfer protein azurin into an active copper enzyme that forms a copper-sulfenate center and demonstrating the importance of noncovalent secondary sphere interactions in stabilizing it constitute important contributions toward the understanding of metal-sulfenate species in biological systems

Toward Engineering Oxygenase Activity into the Electron Transfer Protein Azurin

Structural knowledge of the metal environment is important in understanding the function of metalloproteins and is critical to harnessing their power. Modeling within a protein scaffold offers a pathway to the rational design of desired function s by use of Nature’s own tools; both to gain novel insights into metal-mediated processes and to create new processes. The broad goal of this work is successful engineering of monooxygenase function into model proteinic scaffolds. We seek to model within a prototype scaffold the function of the PHM (peptidylyglycine--hydroxylating monooxygenase) and DβM (dopamine -monooxygenase) family of enzymes, which are capable of remarkable C-H bond activation under ambient conditions with ascorbic acid and molecular oxygen. In Chapter 2, the power of the cavity mutant approach to small molecule processing in the azurin scaffold is demonstrated through description of the first reported Cu(II)-sulfenic acid species. This species, prepared in high yield through copper mediated reduction of hydrogen peroxide, represents the first reported chemical transformation in ‘converted’ electron transfer protein. Furthermore, it is the first report of a synthetic sulfenic acid functionality within the context of a protein. The latter is an important contribution to the field of protein design, as precise knowledge and control of cysteine oxidation state is an important parameter in dictating function in cysteine-containing native and designed peptides and proteins. In Chapter 3, a series of Type-2 azurin variants is reported in which the anionic redox ‘chameleon’ Cys-112 is replaced with neutral His-112 in an effort to develop a robust protein scaffold for modeling of substrate C-H bond activation. A series of surrogate, intramolecular substrates that offer an accessible H-atom were generated through site directed mutagenesis at the axial Met-121 position above the trigonal plane. The spectral characterization of the series is described, and mutant-dependent reactivity with added O2 and ascorbic acid is described. In Chapter 4, the fatty-acid carrier protein, Cellular Retinoic Acid Binding Protein (CRAPB-II) is described in the context of hosting small molecule catalysts. Initial investigations into the design of a binding site for the carbene transfer catalyst, rhodium acetate, will be described, as well as investigation into the utility of the CRABP-II scaffold for aqueous nitric oxide sensing. Finally, in Chapter 5 will be discussed the development of a temperature independent pH buffer, along with demonstration of its utility in low-temperature antibiotic storage, and low temperature spectroscopy