Experimental interrogation of the relationship between protein dynamics and enzyme catalysis is c... more Experimental interrogation of the relationship between protein dynamics and enzyme catalysis is challenging. Light-activated protochlorophyllide oxidoreductase (POR) is an excellent model for investigating this relationship because photoinitiation of the reaction cycle enables coordinated turnover in a "dark-assembled" ternary enzyme-substrate complex. The catalytic cycle involves sequential hydride and proton transfers (from NADPH and an active site tyrosine residue, respectively) to the substrate protochlorophyllide. Studies with a limited cross-species subset of POR enzymes (n = 4) have suggested that protein dynamics associated with hydride and proton transfer are distinct [Heyes, D. J., Levy, C., Sakuma, M., Robertson, D. L., and Scrutton, N. S. (2011) J. Biol. Chem. 286, 11849-11854]. Here, we use steady-state assays and single-turnover laser flash spectroscopy to analyze hydride and proton transfer dynamics in an extended series of POR enzymes taken from many species, including cyanobacteria, algae, embryophytes, and angiosperms. Hydride/proton transfer in all eukaryotic PORs is faster compared to prokaryotic PORs, suggesting active site architecture has been optimized in eukaryotic PORs following endosymbiosis. Visible pump-probe spectroscopy was also used to demonstrate a common photoexcitation mechanism for representative POR enzymes from different branches of the phylogenetic tree. Dynamics associated with hydride transfer are localized to the active site of all POR enzymes and are conserved. However, dynamics associated with proton transfer are variable. Protein dynamics associated with proton transfer are also coupled to solvent dynamics in cyanobacterial PORs, and these networks are likely required to optimize (shorten) the donor-acceptor distance for proton transfer. These extended networks are absent in algal and plant PORs. Our analysis suggests that extended networks of dynamics are disfavored, possibly through natural selection. Implications for the evolution of POR and more generally for other enzyme catalysts are discussed.
The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has be... more The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has been initiated with a 50-fs laser pulse. We show that the catalytic mechanism, involving proton and hydride transfers, proceeds with time constants of 3 ps and 400 ps. It is known that catalysis by POR involves thermally excited protein dynamics; our results show that these molecular motions occur on an ultrafast timescale.
The role of conformational changes in explaining the huge catalytic power of enzymes is currently... more The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology 1-7 . Although it is now widely regarded that enzymes modulate reaction rates by means of short-and long-range protein motions 3-7 , it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers 8 . Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excitedstate lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.
We demonstrate that thiouredopyrene-3,6,8-trisulfonate (TUPS), a photoactivatable reagent, can ra... more We demonstrate that thiouredopyrene-3,6,8-trisulfonate (TUPS), a photoactivatable reagent, can rapidly inject electrons into complex redox enzymes, enabling studies of the kinetics of internal electron that are not accessible using conventional rapid mixing, stopped-flow methods.
Certain bacteria are able to conserve energy via the reductive dehalogenation of halo-organic com... more Certain bacteria are able to conserve energy via the reductive dehalogenation of halo-organic compounds in a respiration-type metabolism. The transcriptional regulator CprK from Desulfitobacterium spp. induces expression of halorespiratory genes upon binding of o-chlorophenol ligands and is reversibly inactivated by oxygen through disulphide bond formation. We report crystal structures of D. hafniense CprK in the ligand-free (both oxidation states), ligand-bound (reduced) and DNA-bound states, making it the first member of the widespread CRP-FNR superfamily for which a complete structural description of both redox-dependent and allosteric molecular rearrangements is available. In conjunction with kinetic and thermodynamic ligand binding studies, we provide a model for the allosteric mechanisms underpinning transcriptional control. Amino acids that play a key role in this mechanism are not conserved in functionally distinct CRP-FNR members. This suggests that, despite significant structural homology, distinct allosteric mechanisms are used, enabling this protein family to control a very wide range of processes.
Journal of the American Chemical Society, Jan 17, 2015
Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen i... more Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen into organic substrates. Typically, they use transition metals or organic cofactors for catalysis. Bacterial 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD) catalyzes the spin-forbidden transfer of dioxygen to its N-heteroaromatic substrate in the absence of any cofactor. We combined kinetics, spectroscopic and computational approaches to establish a novel reaction mechanism. The present work gives insight into the rate limiting steps in the reaction mechanism, the effect of first-coordination sphere amino acids as well as electron-donating/electron-withdrawing substituents on the substrate. We highlight the role of active site residues Ser101/Trp160/His251 and their involvement in the reaction mechanism. The work shows, for the first time, that the reaction is initiated by triplet dioxygen and its binding to deprotonated substrate and only thereafter a spin state crossing to the sing...
The coupling of photochemistry to protein chemical and structural change is crucial to biological... more The coupling of photochemistry to protein chemical and structural change is crucial to biological light-activated signaling mechanisms. This is typified by cyanobacteriochromes (CBCRs), members of the phytochrome superfamily of photoreceptors that exhibit a high degree of spectral diversity, collectively spanning the entire visible spectrum. CBCRs utilize a basic E/Z isomerization of the bilin chromophore as the primary step in their photocycle, which consists of reversible photoconversion between two photostates. Despite intense interest in these photoreceptors as signal transduction modules a complete description of light-activated chemical and structural changes has not been reported. The CBCR Tlr0924 contains both phycocyanobilin and phycoviolobilin chromophores, and these two species photoisomerize in parallel via spectrally and kinetically equivalent intermediates before the second step of the photoreaction where the reaction pathways diverge, the loss of a thioether linkage t...
Journal of the American Chemical Society, Jan 23, 2007
We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid redu... more We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid reduction of the heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochrome P450 BM3. An aqueous electron derived from photoexcited NAD(P)H is rapidly transferred to the heme domain, enabling the formation of a carbon monoxy complex of the ferrous P450 (FeII-CO) on the microsecond time scale. Using this approach we have determined the limiting rate constant (1770 s-1 for substrate-free heme domain) for formation of the FeII-CO complex. We find no dependence of the observed rate of FeII-CO complex formation on NAD(P)H concentration but demonstrate a hyperbolic dependence on carbon monoxide concentration. The apparent dissociation constant for the complex of carbon monoxide bound noncovalently to the ferric form of the BM3 heme domain (and with NADH as reductant) is 323 microM. Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide and ...
The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model sys... more 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. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized 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 attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts.
The reduction of protochlorophyllide (Pchlide) to chlorophyllide, catalysed by the enzyme protoch... more The reduction of protochlorophyllide (Pchlide) to chlorophyllide, catalysed by the enzyme protochlorophyllide oxidoreductase (POR), is the penultimate step in the chlorophyll biosynthetic pathway and is a key light-driven reaction that triggers a profound transformation in plant development. As POR is light-activated it can provide new information on the way in which light energy can be harnessed to power enzyme reactions. Consequently, POR presents a unique opportunity to study catalysis at low temperatures and on ultrafast timescales, which are not usually accessible for the majority of enzymes. Recent advances in our understanding of the catalytic mechanism of POR illustrate why it is an important model for studying enzyme catalysis and reaction dynamics. The reaction involves the addition of one hydride and one proton, and catalysis is initiated by the absorption of light by the Pchlide substrate. As the reaction involves the Pchlide excited state, a variety of ultrafast spectroscopic measurements have shown that significant parts of the reaction occur on the picosecond timescale. A number of excited state Pchlide species, including an intramolecular charge transfer complex and a hydrogen bonded intermediate, are proposed to be required for the subsequent hydride and proton transfers, which occur on the microsecond timescale. Herein, we review spectroscopic investigations, with a particular focus on time-resolved transient absorption and fluorescence experiments that have been used to study the excited state dynamics and catalytic mechanism of POR.
Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of cent... more Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 ?s. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms.
The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has be... more The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has been initiated with a 50-fs laser pulse. We show that the catalytic mechanism, involving proton and hydride transfers, proceeds with time constants of 3 ps and 400 ps. It is known that catalysis by POR involves thermally excited protein dynamics; our results show that these molecular motions occur on an ultrafast timescale.
The (bacterio)chlorophyll biosynthetic pathway is of profound importance to the biosphere. During... more The (bacterio)chlorophyll biosynthetic pathway is of profound importance to the biosphere. During the past 20 years, there have been major advances in the under standing of the genes involved in the pathway and, more recently, in the enzymes that they encode. Chlorophyll biosynthesis can be considered to start with protoporphyrin IX, which lies at the branchpoint with haem synthesis. Therefore,
The temperature dependence of kinetic isotope effects (KIEs) has emerged as the main experimental... more The temperature dependence of kinetic isotope effects (KIEs) has emerged as the main experimental probe of enzymatic H-transfer by quantum tunnelling. Implicit in the interpretation is a presumed role for dynamic coupling of H-transfer chemistry to the protein environment, the so-called 'promoting motions/vibrations hypothesis'. This idea remains contentious, and others have questioned the importance and/or existence of promoting motions/vibrations. New experimental methods of addressing this problem are emerging, including use of mass-modulated enzymes and time-resolved spectroscopy. The pressure dependence of KIEs has been considered as a potential probe of quantum tunnelling reactions, because semi-classical KIEs, which are defined by differences in zero-point vibrational energy, are relatively insensitive to kbar changes in pressure. Reported combined pressure and temperature (p-T) dependence studies of H-transfer reactions are, however, limited. Here, we extend and review the available p-T studies that have utilized well-defined experimental systems in which quantum mechanical tunnelling is established. These include flavoproteins, quinoproteins, light-activated enzymes and chemical model systems. We show that there is no clear general trend between the p-T dependencies of the KIEs in these systems. Given the complex nature of p-T studies, we conclude that computational simulations using determined (e.g. X-ray) structures are also needed alongside experimental measurements of reaction rates/KIEs to guide the interpretation of p-T effects. In providing new insight into H-transfer/environmental coupling, combined approaches that unite both atomistic understanding with experimental rate measurements will require careful evaluation on a case-by-case basis. Although individually informative, we conclude that p-T studies do not provide the more generalized insight that has come from studies of the temperature dependence of KIEs.
In the chlorophyll biosynthetic pathway, the enzyme protochlorophyllide oxidoreductase (POR) cata... more In the chlorophyll biosynthetic pathway, the enzyme protochlorophyllide oxidoreductase (POR) catalyses a key light-driven reaction that triggers a profound transformation in plant development. Because POR is activated by light, it can provide information on the way in which light energy can be harnessed to power enzyme reactions and it presents us with a unique opportunity to study catalysis at low temperatures and on ultrafast timescales that are not accessible for most analyses of enzyme function. Recent advances in our understanding of the catalytic mechanism of POR illustrate why it is an important generic model for studying enzyme catalysis and reaction dynamics.
Protochlorophyllide (PChlide) is a precursor in the biosynthesis of chlorophyll. Complexed with N... more Protochlorophyllide (PChlide) is a precursor in the biosynthesis of chlorophyll. Complexed with NADPH to the enzyme protochlorophyllide oxidoreductase (POR), it is reduced to chlorophyllide, a process that occurs via a set of spectroscopically distinct intermediate states and is initiated from the excited state of PChlide. To obtain a better understanding of these catalytic events, we characterized the excited state dynamics of PChlide in the solvents tetrahydrofuran (THF), methanol, and Tris/Triton buffer using ultrafast transient absorption in the visible and mid-infrared spectral regions and time-resolved fluorescence emission experiments. For comparison, we present time-resolved transient absorption measurements of chlorophyll a in THF. From the combined analysis of these experiments, we derive that during the 2-3 ns excited state lifetime an extensive multiphasic quenching of the emission occurs due to solvation of the excited state, which is in agreement with the previously proposed internal charge transfer (ICT) character of the S1 state (Zhao, G. J.; Han, K. L. . The solvation process in methanol occurs in conjunction with a strengthening of a hydrogen bond to the Pchlide keto carbonyl group. We demonstrate that the internal conversion from the S2 to S1 excited states is remarkably slow and stretches out on to the 700 fs time scale, causing a rise of blue-shifted signals in the transient absorption and a gain of emission in the time-resolved fluorescence. A triplet state is populated on the nanosecond time scale with a maximal yield of ∼23%. The consequences of these observations for the catalytic pathway and the role of the triplet and ICT state in activation of the enzyme are discussed.
The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affini... more The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide (NO) and carbon monoxide (CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O 2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue (R124) and a distal leucine residue (L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124A variant. In the R124A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124A variant of cytochrome c' that is distinct from that in wildtype cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range.
There is a mounting body of evidence to suggest that enzyme motions are linked to function, altho... more There is a mounting body of evidence to suggest that enzyme motions are linked to function, although the design of informative experiments aiming to evaluate how this motion facilitates reaction chemistry is challenging. For the family of diflavin reductase enzymes, typified by cytochrome P450 reductase, accumulating evidence suggests that electron transfer is somehow coupled to large-scale conformational change and that protein motions gate the electron transfer chemistry. These ideas have emerged from a variety of experimental approaches, including structural biology methods (i.e. X-ray crystallography, electron paramagnetic ⁄ NMR spectroscopies and solution X-ray scattering) and advanced spectroscopic techniques that have employed the use of variable pressure kinetic methodologies, together with solvent perturbation studies (i.e. ionic strength, deuteration and viscosity). Here, we offer a personal perspective on the importance of motions to electron transfer in the cytochrome P450 reductase family of enzymes, drawing on the detailed insight that can be obtained by combining these multiple structural and biophysical approaches.
Experimental interrogation of the relationship between protein dynamics and enzyme catalysis is c... more Experimental interrogation of the relationship between protein dynamics and enzyme catalysis is challenging. Light-activated protochlorophyllide oxidoreductase (POR) is an excellent model for investigating this relationship because photoinitiation of the reaction cycle enables coordinated turnover in a "dark-assembled" ternary enzyme-substrate complex. The catalytic cycle involves sequential hydride and proton transfers (from NADPH and an active site tyrosine residue, respectively) to the substrate protochlorophyllide. Studies with a limited cross-species subset of POR enzymes (n = 4) have suggested that protein dynamics associated with hydride and proton transfer are distinct [Heyes, D. J., Levy, C., Sakuma, M., Robertson, D. L., and Scrutton, N. S. (2011) J. Biol. Chem. 286, 11849-11854]. Here, we use steady-state assays and single-turnover laser flash spectroscopy to analyze hydride and proton transfer dynamics in an extended series of POR enzymes taken from many species, including cyanobacteria, algae, embryophytes, and angiosperms. Hydride/proton transfer in all eukaryotic PORs is faster compared to prokaryotic PORs, suggesting active site architecture has been optimized in eukaryotic PORs following endosymbiosis. Visible pump-probe spectroscopy was also used to demonstrate a common photoexcitation mechanism for representative POR enzymes from different branches of the phylogenetic tree. Dynamics associated with hydride transfer are localized to the active site of all POR enzymes and are conserved. However, dynamics associated with proton transfer are variable. Protein dynamics associated with proton transfer are also coupled to solvent dynamics in cyanobacterial PORs, and these networks are likely required to optimize (shorten) the donor-acceptor distance for proton transfer. These extended networks are absent in algal and plant PORs. Our analysis suggests that extended networks of dynamics are disfavored, possibly through natural selection. Implications for the evolution of POR and more generally for other enzyme catalysts are discussed.
The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has be... more The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has been initiated with a 50-fs laser pulse. We show that the catalytic mechanism, involving proton and hydride transfers, proceeds with time constants of 3 ps and 400 ps. It is known that catalysis by POR involves thermally excited protein dynamics; our results show that these molecular motions occur on an ultrafast timescale.
The role of conformational changes in explaining the huge catalytic power of enzymes is currently... more The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology 1-7 . Although it is now widely regarded that enzymes modulate reaction rates by means of short-and long-range protein motions 3-7 , it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers 8 . Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excitedstate lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.
We demonstrate that thiouredopyrene-3,6,8-trisulfonate (TUPS), a photoactivatable reagent, can ra... more We demonstrate that thiouredopyrene-3,6,8-trisulfonate (TUPS), a photoactivatable reagent, can rapidly inject electrons into complex redox enzymes, enabling studies of the kinetics of internal electron that are not accessible using conventional rapid mixing, stopped-flow methods.
Certain bacteria are able to conserve energy via the reductive dehalogenation of halo-organic com... more Certain bacteria are able to conserve energy via the reductive dehalogenation of halo-organic compounds in a respiration-type metabolism. The transcriptional regulator CprK from Desulfitobacterium spp. induces expression of halorespiratory genes upon binding of o-chlorophenol ligands and is reversibly inactivated by oxygen through disulphide bond formation. We report crystal structures of D. hafniense CprK in the ligand-free (both oxidation states), ligand-bound (reduced) and DNA-bound states, making it the first member of the widespread CRP-FNR superfamily for which a complete structural description of both redox-dependent and allosteric molecular rearrangements is available. In conjunction with kinetic and thermodynamic ligand binding studies, we provide a model for the allosteric mechanisms underpinning transcriptional control. Amino acids that play a key role in this mechanism are not conserved in functionally distinct CRP-FNR members. This suggests that, despite significant structural homology, distinct allosteric mechanisms are used, enabling this protein family to control a very wide range of processes.
Journal of the American Chemical Society, Jan 17, 2015
Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen i... more Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen into organic substrates. Typically, they use transition metals or organic cofactors for catalysis. Bacterial 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD) catalyzes the spin-forbidden transfer of dioxygen to its N-heteroaromatic substrate in the absence of any cofactor. We combined kinetics, spectroscopic and computational approaches to establish a novel reaction mechanism. The present work gives insight into the rate limiting steps in the reaction mechanism, the effect of first-coordination sphere amino acids as well as electron-donating/electron-withdrawing substituents on the substrate. We highlight the role of active site residues Ser101/Trp160/His251 and their involvement in the reaction mechanism. The work shows, for the first time, that the reaction is initiated by triplet dioxygen and its binding to deprotonated substrate and only thereafter a spin state crossing to the sing...
The coupling of photochemistry to protein chemical and structural change is crucial to biological... more The coupling of photochemistry to protein chemical and structural change is crucial to biological light-activated signaling mechanisms. This is typified by cyanobacteriochromes (CBCRs), members of the phytochrome superfamily of photoreceptors that exhibit a high degree of spectral diversity, collectively spanning the entire visible spectrum. CBCRs utilize a basic E/Z isomerization of the bilin chromophore as the primary step in their photocycle, which consists of reversible photoconversion between two photostates. Despite intense interest in these photoreceptors as signal transduction modules a complete description of light-activated chemical and structural changes has not been reported. The CBCR Tlr0924 contains both phycocyanobilin and phycoviolobilin chromophores, and these two species photoisomerize in parallel via spectrally and kinetically equivalent intermediates before the second step of the photoreaction where the reaction pathways diverge, the loss of a thioether linkage t...
Journal of the American Chemical Society, Jan 23, 2007
We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid redu... more We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid reduction of the heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochrome P450 BM3. An aqueous electron derived from photoexcited NAD(P)H is rapidly transferred to the heme domain, enabling the formation of a carbon monoxy complex of the ferrous P450 (FeII-CO) on the microsecond time scale. Using this approach we have determined the limiting rate constant (1770 s-1 for substrate-free heme domain) for formation of the FeII-CO complex. We find no dependence of the observed rate of FeII-CO complex formation on NAD(P)H concentration but demonstrate a hyperbolic dependence on carbon monoxide concentration. The apparent dissociation constant for the complex of carbon monoxide bound noncovalently to the ferric form of the BM3 heme domain (and with NADH as reductant) is 323 microM. Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide and ...
The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model sys... more 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. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized 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 attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts.
The reduction of protochlorophyllide (Pchlide) to chlorophyllide, catalysed by the enzyme protoch... more The reduction of protochlorophyllide (Pchlide) to chlorophyllide, catalysed by the enzyme protochlorophyllide oxidoreductase (POR), is the penultimate step in the chlorophyll biosynthetic pathway and is a key light-driven reaction that triggers a profound transformation in plant development. As POR is light-activated it can provide new information on the way in which light energy can be harnessed to power enzyme reactions. Consequently, POR presents a unique opportunity to study catalysis at low temperatures and on ultrafast timescales, which are not usually accessible for the majority of enzymes. Recent advances in our understanding of the catalytic mechanism of POR illustrate why it is an important model for studying enzyme catalysis and reaction dynamics. The reaction involves the addition of one hydride and one proton, and catalysis is initiated by the absorption of light by the Pchlide substrate. As the reaction involves the Pchlide excited state, a variety of ultrafast spectroscopic measurements have shown that significant parts of the reaction occur on the picosecond timescale. A number of excited state Pchlide species, including an intramolecular charge transfer complex and a hydrogen bonded intermediate, are proposed to be required for the subsequent hydride and proton transfers, which occur on the microsecond timescale. Herein, we review spectroscopic investigations, with a particular focus on time-resolved transient absorption and fluorescence experiments that have been used to study the excited state dynamics and catalytic mechanism of POR.
Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of cent... more Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 ?s. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms.
The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has be... more The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has been initiated with a 50-fs laser pulse. We show that the catalytic mechanism, involving proton and hydride transfers, proceeds with time constants of 3 ps and 400 ps. It is known that catalysis by POR involves thermally excited protein dynamics; our results show that these molecular motions occur on an ultrafast timescale.
The (bacterio)chlorophyll biosynthetic pathway is of profound importance to the biosphere. During... more The (bacterio)chlorophyll biosynthetic pathway is of profound importance to the biosphere. During the past 20 years, there have been major advances in the under standing of the genes involved in the pathway and, more recently, in the enzymes that they encode. Chlorophyll biosynthesis can be considered to start with protoporphyrin IX, which lies at the branchpoint with haem synthesis. Therefore,
The temperature dependence of kinetic isotope effects (KIEs) has emerged as the main experimental... more The temperature dependence of kinetic isotope effects (KIEs) has emerged as the main experimental probe of enzymatic H-transfer by quantum tunnelling. Implicit in the interpretation is a presumed role for dynamic coupling of H-transfer chemistry to the protein environment, the so-called 'promoting motions/vibrations hypothesis'. This idea remains contentious, and others have questioned the importance and/or existence of promoting motions/vibrations. New experimental methods of addressing this problem are emerging, including use of mass-modulated enzymes and time-resolved spectroscopy. The pressure dependence of KIEs has been considered as a potential probe of quantum tunnelling reactions, because semi-classical KIEs, which are defined by differences in zero-point vibrational energy, are relatively insensitive to kbar changes in pressure. Reported combined pressure and temperature (p-T) dependence studies of H-transfer reactions are, however, limited. Here, we extend and review the available p-T studies that have utilized well-defined experimental systems in which quantum mechanical tunnelling is established. These include flavoproteins, quinoproteins, light-activated enzymes and chemical model systems. We show that there is no clear general trend between the p-T dependencies of the KIEs in these systems. Given the complex nature of p-T studies, we conclude that computational simulations using determined (e.g. X-ray) structures are also needed alongside experimental measurements of reaction rates/KIEs to guide the interpretation of p-T effects. In providing new insight into H-transfer/environmental coupling, combined approaches that unite both atomistic understanding with experimental rate measurements will require careful evaluation on a case-by-case basis. Although individually informative, we conclude that p-T studies do not provide the more generalized insight that has come from studies of the temperature dependence of KIEs.
In the chlorophyll biosynthetic pathway, the enzyme protochlorophyllide oxidoreductase (POR) cata... more In the chlorophyll biosynthetic pathway, the enzyme protochlorophyllide oxidoreductase (POR) catalyses a key light-driven reaction that triggers a profound transformation in plant development. Because POR is activated by light, it can provide information on the way in which light energy can be harnessed to power enzyme reactions and it presents us with a unique opportunity to study catalysis at low temperatures and on ultrafast timescales that are not accessible for most analyses of enzyme function. Recent advances in our understanding of the catalytic mechanism of POR illustrate why it is an important generic model for studying enzyme catalysis and reaction dynamics.
Protochlorophyllide (PChlide) is a precursor in the biosynthesis of chlorophyll. Complexed with N... more Protochlorophyllide (PChlide) is a precursor in the biosynthesis of chlorophyll. Complexed with NADPH to the enzyme protochlorophyllide oxidoreductase (POR), it is reduced to chlorophyllide, a process that occurs via a set of spectroscopically distinct intermediate states and is initiated from the excited state of PChlide. To obtain a better understanding of these catalytic events, we characterized the excited state dynamics of PChlide in the solvents tetrahydrofuran (THF), methanol, and Tris/Triton buffer using ultrafast transient absorption in the visible and mid-infrared spectral regions and time-resolved fluorescence emission experiments. For comparison, we present time-resolved transient absorption measurements of chlorophyll a in THF. From the combined analysis of these experiments, we derive that during the 2-3 ns excited state lifetime an extensive multiphasic quenching of the emission occurs due to solvation of the excited state, which is in agreement with the previously proposed internal charge transfer (ICT) character of the S1 state (Zhao, G. J.; Han, K. L. . The solvation process in methanol occurs in conjunction with a strengthening of a hydrogen bond to the Pchlide keto carbonyl group. We demonstrate that the internal conversion from the S2 to S1 excited states is remarkably slow and stretches out on to the 700 fs time scale, causing a rise of blue-shifted signals in the transient absorption and a gain of emission in the time-resolved fluorescence. A triplet state is populated on the nanosecond time scale with a maximal yield of ∼23%. The consequences of these observations for the catalytic pathway and the role of the triplet and ICT state in activation of the enzyme are discussed.
The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affini... more The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide (NO) and carbon monoxide (CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O 2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue (R124) and a distal leucine residue (L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124A variant. In the R124A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124A variant of cytochrome c' that is distinct from that in wildtype cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range.
There is a mounting body of evidence to suggest that enzyme motions are linked to function, altho... more There is a mounting body of evidence to suggest that enzyme motions are linked to function, although the design of informative experiments aiming to evaluate how this motion facilitates reaction chemistry is challenging. For the family of diflavin reductase enzymes, typified by cytochrome P450 reductase, accumulating evidence suggests that electron transfer is somehow coupled to large-scale conformational change and that protein motions gate the electron transfer chemistry. These ideas have emerged from a variety of experimental approaches, including structural biology methods (i.e. X-ray crystallography, electron paramagnetic ⁄ NMR spectroscopies and solution X-ray scattering) and advanced spectroscopic techniques that have employed the use of variable pressure kinetic methodologies, together with solvent perturbation studies (i.e. ionic strength, deuteration and viscosity). Here, we offer a personal perspective on the importance of motions to electron transfer in the cytochrome P450 reductase family of enzymes, drawing on the detailed insight that can be obtained by combining these multiple structural and biophysical approaches.
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Papers by Derren Heyes