Iron(IV)hydroxide p K a and the Role of Thiolate Ligation in C–H Bond Activation by Cytochrome P450

213

285

Living organisms have adapted to atmospheric dioxygen by exploiting its oxidizing power while protecting themselves against toxic side effects. Reactive oxygen and nitrogen species formed during oxidative stress, as well as high-potential reactive intermediates formed during enzymatic catalysis, could rapidly and irreversibly damage polypeptides were protective mechanisms not available. Chains of redox-active tyrosine and tryptophan residues can transport potentially damaging oxidizing equivalents (holes) away from fragile active sites and toward protein surfaces where they can be scavenged by cellular reductants. Precise positioning of these chains is required to provide effective protection without inhibiting normal function. A search of the structural database reveals that about one third of all proteins contain Tyr/Trp chains composed of three or more residues. Although these chains are distributed among all enzyme classes, they appear with greatest frequency in the oxidoreductases and hydrolases. Consistent with a redox-protective role, approximately half of the dioxygen-using oxidoreductases have Tyr/Trp chain lengths ≥3 residues. Among the hydrolases, long Tyr/Trp chains appear almost exclusively in the glycoside hydrolases. These chains likely are important for substrate binding and positioning, but a secondary redox role also is a possibility.iron oxygenases | electron transfer | reactive oxygen species | superoxide dismutase | cytochrome P450

Section: Resultsmentioning

confidence: 99%

Hole hopping through tyrosine/tryptophan chains protects proteins from oxidative damage

Gray

2015

213

285

“…Green and coworkers have shown that the compound II intermediates of CYP and CPO are protonated (18,35). CPO-II was estimated to have a pK a ≥ 8.2 based on the fact that the UV-vis spectrum of CPO-II showed no changes from pH 3 to pH 7.…”

Section: Significancementioning

confidence: 99%

“…1B) similar to that of CPO-II. We assign this spectrum to APO-II based on the characteristic split Soret band (370 nm and 428 nm) and two Q bands centered at 535 nm and 567 nm, slightly blue shifted from those of the ferric state (18). APO-I is the dominant intermediate by reacting ferric APO with an oxidant only, such as mCPBA (29,30).…”

Section: Significancementioning

confidence: 99%

“…The central paradigm of CYP oxygen activation is recognized to involve the formation of ferryl intermediates, Fe IV =O. Oxoiron(IV) porphyrin cation radicals (compound I) and oxoiron(IV) porphyrins (compound II) have been observed in CYP (18) as well as classic peroxidases (19), chloroperoxidase (CPO) (20), and catalases (21) and in a variety of model porphyrin systems (22,23). Variations in the reactivities of these ferryl species and the roles of axial ligation and heme environment continue to be vigorously studied and debated.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

Wang

Ullrich

Hofrichter

et al. 2015

A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapidmixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pHjump spectrophotometry revealed a basic pK a of 10.0 for the Fe IV −O−H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C−H and phenolic substrates, with second-order rate constants for C−H and O−H bond scission in the range of 10-10 7 M −1 ·s −1 . Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C−H and O−H bond scission.

“…Although thiolate-ligated compound II species have recently been prepared in both CYP (44) and APO (45) enzymes, these species are produced from the deleterious oxidation of nearby aromatic amino acids from the protein framework (CYP), or by the addition of suitable poised reductants to rapidly quench compound I (APO), rendering them quasi-stable. The catalytic efficiency of the H 2 O 2 single-turnover system (35) and kinetic behavior of the Fe 4+ −OH species observed in OleT demonstrates that Ole-II is a competent reaction intermediate, and that it is directly produced from substrate C−H abstraction.…”

Section: Significancementioning

confidence: 99%

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

Grant

Mitchell

Makris

2016

102

OleT is a cytochrome P450 that catalyzes the hydrogen peroxidedependent metabolism of C n chain-length fatty acids to synthesize C n-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe 4+ −oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C−H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe 4+ −OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon−carbon scission reaction catalyzed by OleT.C ytochrome P450 (CYP) enzymes catalyze an extraordinary breadth of physiologically important oxidations for xenobiotic detoxification and specialized biosynthetic pathways (1, 2). The metabolic diversity of CYP enzymes originates from a sophisticated interplay of substrate molecular recognition with precise tuning of metal oxygen species formed at the enzyme active site. CYPs use a thiolate-ligated heme iron cofactor to activate molecular oxygen and produce short-lived ferric superoxo (3-5), ferric (hydro)peroxo (6-8), and ferryl (9, 10) intermediates. Coordinated efforts over several decades have resulted in isolation of each intermediate, including recent characterization of the principal oxidant thought to be responsible for the vast majority of P450 oxidations, the Fe 4+ −oxo pi−cation radical species commonly referred to as compound I (or P450-I) (10).The archetypal P450 hydroxylation involves C−H bond abstraction by P450-I and ensuing rapid oxygen insertion through a recombination process termed "oxygen rebound" (11, 12). The hydrogen abstraction/rebound mechanism describes the strategy used for most P450 transformations, and is thought to describe catalysis by numerous metal-dependent oxygenases and synthetic bio-inspired metal−oxo complexes (e.g., refs. 13-17). Despite the ubiquity of this mechanism, in some metalloenzymes, a metal−oxo species is formed that is not ultimately destined for incorporation into a substrate. Some characterized examples include the nonheme dinuclear iron ribonucleotide reductase (RNR R2) (18,19) and mononuclear iron halogenase SyrB2 (20-22). The mechanisms for RNR and SyrB2 highlight the importance of quaternary structural elements, an extensive 35-Å proton-coupled electron transfer pathway in RNR (23), and extremely subtle (subangstrom) substrate positional tuning (SyrB2) (21,22), to enable efficient circumvention from the monooxygenation reaction coordinate.P450-I has also been link...