Michiyo Sakuma

Nuclear quantum tunneling in the light-activated enzyme protochlorophyllide oxidoreductase.

In chlorophyll biosynthesis, the light-activated enzyme protochlorophyllide oxidoreductase catalyzes trans addition of hydrogen across the C-17–C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide). This unique light-driven reaction plays a key role in the assembly of the photosynthetic apparatus, but despite its biological importance, the mechanism of light-activated catalysis is unknown. In this study, we show that Pchlide reduction occurs by dynamically coupled nuclear quantum tunneling of a hydride anion followed by a proton on the microsecond time scale in the Pchlide excited and ground states, respectively. We demonstrate the need for fast dynamic searches to form degenerate “tunneling-ready” configurations within the lifetime of the Pchlide excited state from which hydride transfer occurs. Moreover, we have found a breakpoint at -27 °C in the temperature dependence of the hydride transfer rate, which suggests that motions/vibrations that are important for promoting light-activated hydride tunneling are quenched below -27 °C. We observed no such breakpoint for the proton-tunneling reaction, indicating a reliance on different promoting modes for this reaction in the enzyme-substrate complex. Our studies indicate that the overall photoreduction of Pchlide is endothermic and that rapid dynamic searches are required to form distinct tunneling-ready configurations within the lifetime of the photoexcited state. Consequently, we have established the first important link between photochemical and nuclear quantum tunneling reactions, linked to protein dynamics, in a biologically significant system.

A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.

We have conducted a site‐specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β‐unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2‐phenyl‐1‐nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2‐phenylpropanal oxime from 2‐phenyl‐1‐nitropropene. The multiple binding conformations of hydroxy substituted nitro‐olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.

Laser Excitation Studies of the Product Release Steps in the Catalytic Cycle of the Light-driven Enzyme, Protochlorophyllide Oxidoreductase

The latter stages of the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6-ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP+ and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP+ is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s–1. The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP+ concentration, suggesting that conformational changes are required prior to NADP+ release. Following NADP+ release, the NADPH rebinds to the enzyme with a maximum rate constant of ∼72 s–1. At elevated temperatures (>298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s–1. The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.

Active site modifications in pentaerythritol tetranitrate reductase can lead to improved product enantiopurity, decreased by-product formation and altered stereochemical outcome in reactions with α,β-unsaturated nitroolefins

This work describes a site-directed mutagenesis study of pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of key active site residues in influencing both product enantiopurity and the ratio of CC vs. nitro-group reduction with 2-phenyl-1-nitropropene. Comparative biotransformations of wild type and single/double mutants of PETN reductase with 2-phenyl-1-nitropropene showed that one enzyme scaffold was capable of generating both enantiomeric products with improved enantiopurities by a manipulation of the reaction conditions and/or the presence of a one or two key mutations. These changes located at key active site residues were sufficient to moderately improve product enantiopurity, cause a switch in the major product enantiomer formed and/or promote or eliminate side-product formation. The mutation of substrate-binding residue Y351 to alanine and phenylalanine improved the biocatalytic potential of PETN reductase by the elimination of a competing side reaction. The crystal structures of three mutants at residue Y351 (PDB codes: 3P81, 3P84 and 3P8J) show that only subtle changes in the active site environment may be necessary to generate significantly improved biocatalysts.

Human cryptochrome-1 confers light independent biological activity in transgenic drosophila correlated with flavin radical stability

Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.

Solvent‐Slaved Protein Motions Accompany Proton but Not Hydride Tunneling in Light‐Activated Protochlorophyllide Oxidoreductase

H(+) but not H(-): The reduction reaction of protochlorophyllide catalyzed by protochlorophyllide oxidoreductase features solvent-slaved motions that control the proton- but not the hydride-tunneling mechanism. These motions imply a long-range dynamic network from the solvent to the enzyme active site that facilitate proton transfer (see picture, left). Motions for hydride transfer are more localized and are not slaved by the solvent (see picture, right).

A Twin-track Approach Has Optimized Proton and Hydride Transfer by Dynamically Coupled Tunneling during the Evolution of Protochlorophyllide Oxidoreductase

Protein dynamics are crucial for realizing the catalytic power of enzymes, but how enzymes have evolved to achieve catalysis is unknown. The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes sequential hydride and proton transfers in the photoexcited and ground states, respectively, and is an excellent system for relating the effects of motions to catalysis. Here, we have used the temperature dependence of isotope effects and solvent viscosity measurements to analyze the dynamics coupled to the hydride and proton transfer steps in three cyanobacterial PORs and a related plant enzyme. We have related the dynamic profiles of each enzyme to their evolutionary origin. Motions coupled to light-driven hydride transfer are conserved across all POR enzymes, but those linked to thermally activated proton transfer are variable. Cyanobacterial PORs require complex and solvent-coupled dynamic networks to optimize the proton donor-acceptor distance, but evolutionary pressures appear to have minimized such networks in plant PORs. POR from Gloeobacter violaceus has features of both the cyanobacterial and plant enzymes, suggesting that the dynamic properties have been optimized during the evolution of POR. We infer that the differing trajectories in optimizing a catalytic structure are related to the stringency of the chemistry catalyzed and define a functional adaptation in which active site chemistry is protected from the dynamic effects of distal mutations that might otherwise impact negatively on enzyme catalysis.

Ultrafast red light activation of Synechocystis phytochrome Cph1 triggers major structural change to form the Pfr signalling-competent state.

Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel ‘head-to-head’ arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.

Photochemical mechanism of an atypical algal phytochrome

Phytochromes are bilin‐containing photoreceptors that are typically sensitive to the red/far‐red region of the visible spectrum. Recently, phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Herein, a combination of time‐resolved spectroscopy and structural approaches across picosecond to second timescales have been used to map photochemical mechanisms and structural changes in this atypical group of phytochromes. The photochemistry of an orange/far‐red light‐sensitive algal phytochrome from Dolihomastix tenuilepis has been investigated by using a combination of visible, IR and X‐ray scattering probes. The entire photocycle, correlated with accompanying structural changes in the cofactor/protein, are reported. This study identifies a complex photocycle for this atypical phytochrome. It also highlights a need to combine outcomes from a range of biophysical approaches to unravel complex photochemical and macromolecular processes in multi‐domain photoreceptor proteins that are the basis of biological light‐mediated signalling.