Nuclear Quantum Tunneling in the Light-activated Enzyme Protochlorophyllide Oxidoreductase

Heyes, Derren J.; Sakuma, Michiyo; Visser, Sam P. de; Scrutton, Nigel S.

doi:10.1074/jbc.m808548200

Cited by 86 publications

(230 citation statements)

References 38 publications

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“…This second means of reaching tunneling-ready distances has been invoked in the context of the repeated observation of temperature-independent kinetic isotope effects for enzymes functioning under their physiological conditions and with their preferred substrates (15,16). Impaired enzyme function in the presence of modifications distal to the active site (16) or at cryogenic temperatures has previously been attributed to the disruption of functional conformational sampling (27,43). In the present work, we argue that anomalous Arrhenius preexponential factors provide a previously unrecognized probe of the role of conformational sampling in enzyme reactions.…”

Section: Discussionmentioning

confidence: 99%

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Nagel

Dong

Bahnson

et al. 2011

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

152

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A growing body of data supports a role for protein motion in enzyme catalysis. In particular, the ability of enzymes to sample catalytically relevant conformational substates has been invoked to model kinetic and spectroscopic data. However, direct experimental links between rapidly interconverting conformations and the chemical steps of catalysis remain rare. We report here on the kinetic analysis and characterization of the hydride transfer step catalyzed by a series of mutant thermophilic alcohol dehydrogenases (ht-ADH), presenting evidence for Arrhenius prefactor values that become enormously elevated above an expected value of approximately 10 13 s −1 when the enzyme operates below its optimal temperature range. Restoration of normal Arrhenius behavior in the ht-ADH reaction occurs at elevated temperatures. A simple model, in which reduced temperature alters the ability of the ht-ADH variants to sample the catalytically relevant region of conformational space, can reproduce the available data. These findings indicate an impaired landscape that has been generated by the combined condition of reduced temperature and mutation at a single, active-site hydrophobic side chain. The broader implication is that optimal enzyme function requires the maintenance of a relatively smooth landscape that minimizes low energy traps.A complete understanding of the origins of the remarkable rate acceleration and specificity achieved by enzymes remains elusive. Increasingly, studies on the fundamental basis for enzymatic catalysis have focused on the requirement for a range of protein motions in order to achieve efficient enzyme catalysis. Among these, evidence for a class of motion termed conformational sampling (or preorganization) has begun to accumulate recently (cf. refs. 1-4). The impact of such motions can be seen either in the formation of an enzyme-substrate complex or in its subsequent conversion to product.For example, a recent study of adenylate kinase implicates a temperature-dependent preequilibrium among conformers that are either "competent" or "incompetent" toward ligand binding (5). In the context of catalysis, studies of single-enzyme molecules indicate multiple, slowly interconverting conformers that lead to product with different rate constants (6-8). Extensive work on dihydrofolate reductase has shown the role of slow loop closures that occur along the reaction coordinate with the same (millsecond) time constants as catalysis (9, 10).Although computational studies also suggest a role for rapid conformational sampling in enzyme reactions (11, 12), demonstrating the direct link of these motions to catalysis is quite challenging. In this study, we use the thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) as a system to examine the presence of rapidly interconverting protein substates during catalysis. The ht-ADH catalyzes the transfer of a hydride equivalent from an alcohol donor to an NAD þ cofactor, and previous studies have shown that ht-ADH carries out its reaction via quantum t...

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Section: Discussionmentioning

confidence: 99%

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Nagel

Dong

Bahnson

et al. 2011

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

152

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“…Kinetic isotope effect (KIE) for the conversion of ketones into phenols was calculated by replacing the transferring hydrogen atom or the aromatic hydrogen atoms by deuterium atoms in the substrate following procedures described previously [42,43]. Initial semi-classical KIEs were estimated from the Eyring equation using the free energies of activation (G ‡ H ) of the deuterium substituted and reference systems, Eq 1, with T the estimated temperature (298 K) and R the gas constant.…”

Section: The Studies Presented In This Work Use Density Functional Thmentioning

confidence: 99%

Arene activation by a nonheme iron(III)–hydroperoxo complex: pathways leading to phenol and ketone products

2016

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Iron(III)-hydroperoxo complexes are found in various nonheme iron enzymes as catalytic cycle intermediates; however, little is known on their catalytic properties. Recent work of Banse and coworkers on a biomimetic nonheme iron(III)-hydroperoxo complex provided evidence of its involvement in reactivity with arenes. This contrasts the behavior of heme iron(III)-hydroperoxo complexes that are known to be sluggish oxidants. In order to gain insight into the reaction mechanism of the biomimetic iron(III)-hydroperoxo complex with arenes we performed a computational (density functional theory) study. The calculations show that iron(III)-hydroperoxo reacts with substrates via low free energies of activation that should be accessible at room temperature. Moreover, dominant ketone reaction product is observed as primary products rather than the thermodynamically more stable phenols. These product distributions are analyzed and the calculations show that charge interaction between the iron(III)-hydroxo group and the substrate in the intermediate state pushes the transferring proton to the metacarbon atom of substrate and guides the selectivity of ketone formation. These studies show that the relative ratio of ketone versus phenol as primary products can be affected by external interactions of the oxidant with substrate. Moreover, iron(III)-hydroperoxo complexes are shown to selectively give ketone products, whereas iron(IV)-oxo complexes will react with arenes to form phenols instead.

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“…The hydride and proton transfer reactions occur in as equential mechanism on the microsecond timescale by nuclear tunneling. [6] Catalysis by POR is also dependent on picosecond excited-state processes within the Pchlide substrate itself. [3] An umber of time-resolved transient spectroscopy studies have identified different short-lived Pchlide* species,both in the isolated pigment [11][12][13][14][15] and in the ternary enzyme-substrate complex.…”

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confidence: 99%

“…Consequently,P OR has become an important model system for studying many aspects of enzyme catalysis. [2][3][4][5][6][7][8][9] Ther eaction catalyzed by POR involves ah ighly endergonic light-driven hydride transfer from the pro-S face of the nicotinamide ring of NADPH to the C17 position of the Pchlide molecule, [5,6] followed by an exergonic thermally activated proton transfer, most likely from ac onserved Tyr residue,t ot he C18 position of Pchlide [9] (Figure 1). The hydride and proton transfer reactions occur in as equential mechanism on the microsecond timescale by nuclear tunneling.…”

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confidence: 99%

Excited‐State Charge Separation in the Photochemical Mechanism of the Light‐Driven Enzyme Protochlorophyllide Oxidoreductase

et al. 2014

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The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions.T he ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride-and proton-transfer chemistry,h ave so far proven difficult to detect. We have used ac ombination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond-microsecond time range,t op ropose an ew mechanism for the photochemistry. Excited-state interactions between active site residues and acarboxyl group on the Pchlide molecule result in apolarized and highly reactive double bond. This so-called "reactive" intramolecular charge-transfer state creates an electron-deficient site across the double bond to trigger the subsequent nucleophilic attacko fN ADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excitedstate processes and chemistry in POR. Moreover,i tp rovides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of lightactivated chemical and biological catalysts.

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Nuclear Quantum Tunneling in the Light-activated Enzyme Protochlorophyllide Oxidoreductase

Cited by 86 publications

References 38 publications

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Impaired protein conformational landscapes as revealed in anomalous Arrhenius prefactors

Arene activation by a nonheme iron(III)–hydroperoxo complex: pathways leading to phenol and ketone products

Excited‐State Charge Separation in the Photochemical Mechanism of the Light‐Driven Enzyme Protochlorophyllide Oxidoreductase

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