Modulation of ligand-heme reactivity by binding pocket residues demonstrated in cytochrome c' over the femtosecond-second temporal range

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 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 wild-type 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. © 2013 FEBS.

A microbial platform for renewable propane synthesis based on a fermentative butanol pathway

Background Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli. Results We report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 μg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L). Conclusions This study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde

Photochemical Spin Dynamics of the Vitamin B12 Derivative, Methylcobalamin

Derivatives of vitamin B12 are six-coordinate cobalt corrinoids found in humans, other animals and micro-organisms. By acting as enzymatic cofactors and photoreceptor chromophores they serve vital metabolic and photoprotective functions. Depending on the context, the chemical mechanisms of the biologically-active derivatives of B12 – methylcobalamin (MeCbl) and 5’-deoxyadenosylcobalamin (AdoCbl) – can be very different from one another. The extent to which this chemistry is tuned by the upper axial ligand, however, is not yet clear. Here, we have used a combination of time-resolved FT-EPR, magnetic field effect experiments and spin dynamic simulations to reveal that the upper axial ligand alone only results in relatively minor changes to the photochemical spin dynamics of B12. By studying the photolysis of MeCbl, we find that, much like for AdoCbl, the initial (or ‘geminate’) radical pairs are born predominantly in the singlet spin-state and thus originate from singlet excited-state precursors. This is in contrast to the triplet radical pairs and precursors proposed previously. Unlike AdoCbl, the extent of geminate recombination is limited following MeCbl photolysis, resulting in significant distortions to the FT-EPR signal caused by polarization from spin-correlated methyl-methyl radical ‘f-pairs’ formed following rapid diffusion. Despite the photophysical mechanism that precedes photolysis of MeCbl showing a wavelength-dependence, the subsequent spin dynamics appear to be largely independent of excitation wavelength, again much like for AdoCbl. Our data finally provide clarity to what in the literature to date has been a confused and contradictory picture. We conclude that, although the upper axial position of MeCbl and AdoCbl does impact their reactivity to some extent, the remarkable biochemical diversity of these fascinating molecules is most likely a result of tuning by their protein environment

Direct Evidence of an Excited-State Triplet Species upon Photoactivation of the Chlorophyll Precursor Protochlorophyllide

The chlorophyll precursor protochlorophyllide (Pchlide), which is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase, has unique excited-state properties that facilitate photocatalysis. Previous time-resolved spectroscopy measurements have implied that a long-lived triplet state is formed during the excited-state relaxation of Pchlide, although direct evidence of its existence is still lacking. Here we use time-resolved electron paramagnetic resonance (EPR) in combination with time-resolved absorption measurements at a range of temperatures (10–290 K), solvents, and oxygen concentrations to provide a detailed characterization of the triplet state of Pchlide. The triplet decays in a biphasic, oxygen-dependent manner, while the first reported EPR signature of a Pchlide triplet displays both emissive and absorptive features and an antisymmetric spectrum similar to other porphyrin triplet states. This work demonstrates that the Pchlide triplet is accessible to various cryogenic spectroscopic probes over a range of time scales and paves the way for understanding its potential role in catalysis

Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis

© 2019 American Chemical Society. It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models

Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison of three aldehyde reductase/alcohol dehydrogenase genes

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we have compared the effect of various alcohol dehydrogenases (ADH) for the last step of the isobutanol production. E. coli has the yqhD gene which encodes a broad-range ADH. Isobutanol production significantly decreased with the deletion of yqhD, suggesting that the yqhD gene on the genome contributed to isobutanol production. The adh genes of two bacteria and one yeast were also compared in E. coli harboring the isobutanol synthesis pathway. Overexpression of yqhD or adhA in E. coli showed better production than ADH2, a result confirmed by activity measurements with isobutyraldehyde

Structural basis of catalysis in the bacterial monoterpene synthases linalool synthase and 1,8-cineole synthase

Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids

Quantum biology: an update and perspective

This is the final version. Available from MDPI via the DOI in this record. Data Availability Statement: Not applicable.Understanding the rules of life is one of the most important scientific endeavours and has revolutionised both biology and biotechnology. Remarkable advances in observation tech-niques allow us to investigate a broad range of complex and dynamic biological processes in which living systems could exploit quantum behaviour to enhance and regulate biological functions. Recent evidence suggests that these non-trivial quantum mechanical effects may play a crucial role in maintaining the non-equilibrium state of biomolecular systems. Quantum biology is the study of such quantum aspects of living systems. In this review, we summarise the latest progress in quantum biology, including the areas of enzyme-catalysed reactions, photosynthesis, spin-dependent reactions, DNA, fluorescent proteins, and ion channels. Many of these results are expected to be fundamental building blocks towards understanding the rules of life.Leverhulme Trus