Tryptophan‐96 in cytochrome <scp>P450 BM3</scp> plays a key role in enzyme survival

Ravanfar, Raheleh; Sheng, Yuling; Gray, Harry B.; Winkler, Jay R.

doi:10.1002/1873-3468.14514

Cited by 7 publications

(10 citation statements)

References 21 publications

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“…1 ) produce superoxide, hydrogen peroxide, or water instead of hydroxylated substrate. We have suggested that electron transport chains in P450 composed of tryptophan (W) and tyrosine (Y) residues participate in the water-producing oxidase shunt to protect the enzyme from damage when substrate hydroxylation is impaired ( 16 – 21 ).…”

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confidence: 99%

Tryptophan extends the life of cytochrome P450

Ravanfar,

Sheng,

Gray

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Powerfully oxidizing enzymes need protective mechanisms to prevent self-destruction. The flavocytochrome P450 BM3 from Priestia megaterium (P450 BM3 ) is a self-sufficient monooxygenase that hydroxylates fatty acid substrates using O 2 and NADPH as co-substrates. Hydroxylation of long-chain fatty acids (≥C 14 ) is well coupled to O 2 and NADPH consumption, but shorter chains (≤C 12 ) are more poorly coupled. Hydroxylation of p -nitrophenoxydodecanoic acid by P450 BM3 produces a spectrophotometrically detectable product wherein the coupling of NADPH consumption to product formation is just 10%. Moreover, the rate of NADPH consumption is 1.8 times that of O 2 consumption, indicating that an oxidase uncoupling pathway is operative. Measurements of the total number of enzyme turnovers before inactivation (TTN) indicate that higher NADPH concentrations increase TTN. At lower NADPH levels, added ascorbate increases TTN, while a W96H mutation leads to a decrease. The W96 residue is about 7 Å from the P450 BM3 heme and serves as a gateway residue in a tryptophan/tyrosine (W/Y) hole transport chain from the heme to a surface tyrosine residue. The data indicate that two oxidase pathways protect the enzyme from damage by intercepting the powerfully oxidizing enzyme intermediate (Compound I) and returning it to its resting state. At high NADPH concentrations, reducing equivalents from the flavoprotein are delivered to Compound I by the usual reductase pathway. When NADPH is not abundant, however, oxidizing equivalents from Compound I can traverse a W/Y chain, arriving at the enzyme surface where they are scavenged by reductants. Ubiquitous tryptophan/tyrosine chains in highly oxidizing enzymes likely perform similar protective functions.

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confidence: 99%

Tryptophan extends the life of cytochrome P450

Ravanfar,

Sheng,

Gray

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, certain protective mechanisms must exist to protect against oxidative damage. One possible mechanism, proposed by Gray and Winkler, − suggests that successive electron transfer reactions through a chain of redox-active residues (like tryptophan, methionine, and tyrosine) could eventually deliver the radical to the protein’s surface. This action would defuse the ‘redox bomb’ at the catalytic center .…”

Section: Discussionmentioning

confidence: 99%

Understanding the Multifaceted Mechanism of Compound I Formation in Unspecific Peroxygenases through Multiscale Simulations

Costa,

Liang

2023

J. Phys. Chem. B

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Unspecific peroxygenases (UPOs) can selectively oxyfunctionalize unactivated hydrocarbons by using peroxides under mild conditions. They circumvent the oxygen dilemma faced by cytochrome P450s and exhibit greater stability than the latter. As such, they hold great potential for industrial applications. A thorough understanding of their catalysis is needed to improve their catalytic performance. However, it remains elusive how UPOs effectively convert peroxide to Compound I (CpdI), the principal oxidizing intermediate in the catalytic cycle. Previous computational studies of this process primarily focused on heme peroxidases and P450s, which have significant differences in the active site from UPOs. Additionally, the roles of peroxide unbinding in the kinetics of CpdI formation, which is essential for interpreting existing experiments, have been understudied. Moreover, there has been a lack of free energy characterizations with explicit sampling of protein and hydration dynamics, which is critical for understanding the thermodynamics of the proton transport (PT) events involved in CpdI formation. To bridge these gaps, we employed multiscale simulations to comprehensively characterize the CpdI formation in wild-type UPO from Agrocybe aegerita (AaeUPO). Extensive free energy and potential energy calculations were performed in a quantum mechanics/molecular mechanics setting. Our results indicate that substrate-binding dehydrates the active site, impeding the PT from H2O2 to a nearby catalytic base (Glu196). Furthermore, the PT is coupled with considerable hydrogen bond network rearrangements near the active site, facilitating subsequent O–O bond cleavage. Finally, large unbinding free energy barriers kinetically stabilize H2O2 at the active site. These findings reveal a delicate balance among PT, hydration dynamics, hydrogen bond rearrangement, and cosubstrate unbinding, which collectively enable efficient CpdI formation. Our simulation results are consistent with kinetic measurements and offer new insights into the CpdI formation mechanism at atomic-level details, which can potentially aid the design of next-generation biocatalysts for sustainable chemical transformations of feedstocks.

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“…Rosetta simulations identified mutation to Phe as the lowest energy solution. While replacing Trp with Phe could still allow aromatic ring coupling with W178 in theory, previous studies have shown that Phe impedes transference of holes relative to Trp [57]- [59]. Fig.…”

Section: Mapping Of the Tryptophanyl Featurementioning

confidence: 97%

Mutational dissection of a hole hopping route that can be tuned to boost peroxygenase activity in LPMOs

Eijsink,

Ayuso-Fernández,

Emrich-Mills

et al. 2023

Preprint

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Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial lytic polysaccharide monooxygenase (LPMO), member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a Tyr-His pair in the protein’s surface. Real-time monitoring of radical formation revealed a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a mutant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

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Tryptophan‐96 in cytochrome P450 BM3 plays a key role in enzyme survival

Cited by 7 publications

References 21 publications

Tryptophan extends the life of cytochrome P450

Tryptophan extends the life of cytochrome P450

Understanding the Multifaceted Mechanism of Compound I Formation in Unspecific Peroxygenases through Multiscale Simulations

Mutational dissection of a hole hopping route that can be tuned to boost peroxygenase activity in LPMOs

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