Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.

Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs).

Solution-State Inter-Copper Distribution of Redox Partner-Linked Copper Nitrite Reductases: A Pulsed Electron-Electron Double Resonance Spectroscopy Study.

Copper nitrite reductases (CuNiRs) catalyze the reduction of nitrite to form nitric oxide. In recent years, new classes of redox partner linked CuNiRs have been isolated and characterized by crystallographic techniques. Solution-state biophysical studies have shed light on the complex catalytic mechanisms of these enzymes and implied that protein dynamics may play a role in CuNiR catalysis.

Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases.

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme.

Insights into the H O -driven catalytic mechanism of fungal lytic polysaccharide monooxygenases.

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H O is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H O is therefore of importance for their use in biotechnological settings.

Photochemical Mechanism of Light-Driven Fatty Acid Photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a promising target for the production of biofuels and fine chemicals. It contains a flavin adenine dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymatic decarboxylation.

Protein Conformational Change Is Essential for Reductive Activation of Lytic Polysaccharide Monooxygenase by Cellobiose Dehydrogenase.

Large-scale protein domain dynamics and electron transfer are often associated. However, as protein motions span a broad range of time and length scales, it is often challenging to identify and thus link functionally relevant dynamic changes to electron transfer in proteins. It is hypothesized that large-scale domain motions direct electrons through a FAD and a heme cofactor of the fungal cellobiose dehydrogenase (CDH) enzymes to the type-II copper center (T2Cu) of the polysaccharide-degrading lytic polysaccharide monooxygenases (LPMOs).

Active Intermediates in Copper Nitrite Reductase Reactions Probed by a Cryotrapping-Electron Paramagnetic Resonance Approach.

Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes.

Radical-based photoinactivation of fatty acid photodecarboxylases.

Fatty acid photodecarboxylases (FAP) are a recently discovered family of FAD-containing, light-activated enzymes, which convert fatty acids to n-alkanes/alkenes with potential applications in the manufacture of fine and speciality chemicals and fuels. Poor catalytic stability of FAPs is however a major limitation. Here, we describe a methodology to purify catalytically stable and homogeneous samples of recombinant Chlorella variabilis NC64A FAP (CvFAP) from Escherichia coli.

Solvent-slaved protein motions accompany proton coupled electron transfer reactions catalysed by copper nitrite reductase.

Through the use of time-resolved pH-jump spectroscopy, we demonstrate how proton transfer is coupled to inter-copper electron transfer in a copper nitrite reductase (CuNiR). Combined use of electron paramagnetic resonance spectroscopy with solvent viscosity- and pressure-dependence pH-jump stopped-flow spectroscopy is used to show that solvent-slaved protein motions are linked to this proton coupled electron transfer step in CuNiR.

Selectivity through discriminatory induced fit enables switching of NAD(P)H coenzyme specificity in Old Yellow Enzyme ene-reductases.

Most ene-reductases belong to the Old Yellow Enzyme (OYE) family of flavin-dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron-withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs.

Tripping the light fantastic in membrane redox biology: linking dynamic structures to function in ER electron transfer chains.

How the dynamics of proteins assist catalysis is a contemporary issue in enzymology. In particular, this holds true for membrane-bound enzymes, where multiple structural, spectroscopic and biochemical approaches are needed to build up a comprehensive picture of how dynamics influence enzyme reaction cycles. Of note are the recent studies of cytochrome P450 reductases (CPR)-P450 (CYP) endoplasmic reticulum redox chains, showing the relationship between dynamics and electron flow through flavin and haem redox centres and the impact this has on monooxygenation chemistry.

Trapping methods for probing functional intermediates in nitric oxide synthases and related enzymes.

The three mammalian isoforms of nitric oxide synthase (NOS) produce the signalling molecule nitric oxide (NO) from L-arginine, molecular oxygen, and NADPH. NOS-generated NO is essential for many key biochemical processes and aberrant NO production is linked to the pathophysiology of many diseases. Over the past 40 years, the mechanism and structure of NOS have been studied extensively and there are many quality reviews and perspectives documenting the current hypotheses in the field.

A perspective on conformational control of electron transfer in nitric oxide synthases.

This perspective reviews single molecule and ensemble fluorescence spectroscopy studies of the three tissue specific nitric oxide synthase (NOS) isoenzymes and the related diflavin oxidoreductase cytochrome P450 reductase. The focus is on the role of protein dynamics and the protein conformational landscape and we discuss how recent fluorescence-based studies have helped in illustrating how the nature of the NOS conformational landscape relates to enzyme turnover and catalysis.

Correlating Calmodulin Landscapes with Chemical Catalysis in Neuronal Nitric Oxide Synthase using Time-Resolved FRET and a 5-Deazaflavin Thermodynamic Trap.

A major challenge in enzymology is the need to correlate the dynamic properties of enzymes with, and understand the impact on, their catalytic cycles. This is especially the case with large, multicenter enzymes such as the nitric oxide synthases (NOSs), where the importance of dynamics has been inferred from a variety of structural, single-molecule, and ensemble spectroscopic approaches but where motions have not been correlated experimentally with mechanistic steps in the reaction cycle. Here we take such an approach.

Real-time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c.

Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through an inferred dynamic cycle of 'open' and 'closed' conformations of the enzyme (PLoS Biol, 2011, e1001222). However, these studies were restricted to stopped-flow/FRET analysis of the reductive half-reaction, and were compromised by fluorescence quenching of the acceptor by the flavin cofactors.

Excited-state charge separation in the photochemical mechanism of the light-driven enzyme protochlorophyllide oxidoreductase.

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. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride- and proton-transfer chemistry, have so far proven difficult to detect. We have used a combination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond-microsecond time range, to propose a new mechanism for the photochemistry.