Copper–sulfenate complex from oxidation of a cavity mutant ofPseudomonas aeruginosaazurin

Abstract

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 g z 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.bioinorganic chemistry | metalloprotein design | protein engineering | blue copper proteins | posttranslational modification T he presence of cysteine sulfenic acid (Cys-SOH) as a product of posttranslational oxidation of the cysteine thiol side chain has been found to play important roles in biology (1-6) in the context of metal coordination (7), enzyme-protein regulation (8, 9), redox signaling (1, 5), and gene regulation (10-13). Unlike its higher oxidation state counterparts, i.e., sulfinic (Cys-SO 2 ) and sulfonic (Cys-SO 3 ) acids, the sulfenic acid is inherently unstable and highly reactive and thus requires stabilization to operate in a controlled context in biological systems (1-5). For example, a crystal structure of a sulfenic acid form of SarZ, a redox active global transcriptional regulator in Staphylococcus aureus, revealed that cysteine sulfenic acid is stabilized through two hydrogen bonds with surrounding residues, and its reversible oxidation-reduction allows redox-mediated virulence regulation in S. aureus (13).Because of the inherent instability and high reactivity, few examples of cysteine sulfenic acid have been observed in metalbinding sites in proteins (7,14). Nature has evolved proteins to allow metal ions to coordinate sulfenic acids, resulting in interesting functions. For example, nitrile hydratase, a metalloenzyme which has long found utility in the industrial synthesis of acrylamide (15...

Reversible S-nitrosylation in an engineered azurin

“… 35 Construction, expression, and purification of apo-M121H/H46EAz was carried out following a reported procedure. 28 Titration of apo-M121H/H46EAz with CuSO 4 saturated at 1 equivalent Cu(II). The resulting Cu(II)-M121H/H46EAz showed a strong absorption band at 403 nm (ε = 2300 M -1 cm -1 ) and a weak and broad band around 750 nm ( Supplementary Figs 2 and 3 ) distinct from that of WTAz, but similar to that of nitrosocyanin.…”

Electrochemical, Spectroscopic, and Density Functional Theory Characterization of Redox Activity in Nickel-Substituted Azurin: A Model for Acetyl-CoA Synthase

“…22 The delocalization and large orbital contribution from sulfur highlight the covalency of the metal−thiolate bond and indicate potential for ligand-centered reactivity, as has been seen in mutants of CuAz. 77 Some differences are evident, however, between Cu II Az and Ni I Az. Cu II Az remains in a pseudotetragonal coordination environment in both the Cu II and Cu I redox states, having weak interactions with the glycine carbonyl and methionine axial ligands, while Ni I Az appears to lose both of those axially coordinated ligands upon reduction, giving a trigonal planar species.…”

DNA mismatch repair and oligonucleotide end-protection promote base-pair substitution distal from a CRISPR/Cas9-induced DNA break

“…In contrast, ample evidence exists (our present work, refs. 8–10 ) that during double-stranded break repair, the oligonucleotide only serves as a template and does not physically integrates into the genome. It is therefore conceivable that PTO-containing ssODNs only confer toxicity when they become introduced into the genome.…”

“…The template model predicts that only mutations encoded within the 3′ half of the ssODN will result in mismatched DNA, as this is the only region that can anneal to a 3′ gDNA end. More recently, models reminiscent of the bridge and template models have been proposed for ssODN-mediated repair of DNA nicks, dependent on the polarity of the ssODN with respect to the nicked strand ( 8–10 ). A ssODN complementary to the intact strand (‘cI donor’) would effectuate repair and concomitant gene modification via ‘single-strand DNA incorporation’ (ssDI), while an ssODN complementary to the nicked strand (‘cN donor’) would serve as a template for DNA synthesis in a process referred to as ‘synthesis-dependent strand annealing’ (SDSA) ( 10 ), or perhaps more accurately ‘annealing-driven strand synthesis’ (ADSS) ( 9 ).…”