Recombinant Acetyl-CoA decarbonylase/synthase complex subunit beta 1 (cdhC1)

What is the Acetyl-CoA decarbonylase/synthase complex and where is it found?

Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex found predominantly in methanogens and certain other Archaea. This complex plays a crucial role in the carbon metabolism of these organisms by catalyzing the reversible synthesis and cleavage of the acetyl C−C and C−S bonds of acetyl-CoA. The reaction is integral to both autotrophic carbon fixation and methanogenesis from acetate, representing key metabolic pathways in these microorganisms .

The enzyme complex contains several subunits with distinct functions, with the beta subunit (cdhC1) housing the catalytically essential A-cluster that serves as the active site for the acetyl-CoA synthesis reaction. This subunit has received significant research attention due to its unique metal center composition and catalytic properties .

What is the structure and composition of the A-cluster in the beta subunit?

The A-cluster is a unique metallocofactor located on the beta subunit (cdhC1) of the ACDS complex. Its structure consists of a binuclear Ni−Ni site that is bridged by a cysteine thiolate to an Fe₄S₄ center . This distinctive arrangement creates a specialized active site environment that enables the complex chemistry involved in acetyl-CoA synthesis and cleavage.

The spatial organization of this metal center is critical for its function, as it provides the necessary electronic and spatial properties to facilitate the carbon-carbon and carbon-sulfur bond manipulations required for the reaction. The proximity of the nickel centers to the iron-sulfur cluster allows for effective electron transfer during catalysis, which is essential for the redox chemistry that occurs during the reaction mechanism .

How does the ACDS beta subunit contribute to acetyl-CoA synthesis?

The beta subunit (cdhC1) of ACDS contains the A-cluster active site where acetyl-CoA synthesis occurs. The synthesis reaction involves the combination of a methyl group (derived from methylated corrinoid proteins in vivo, or methylcobinamide in experimental settings), carbon monoxide (CO), and coenzyme A (CoA) .

In experimental conditions, researchers have achieved high rates of acetyl-CoA synthesis using recombinant ACDS beta subunit with methylcobinamide as a mimic of the physiological base-off corrinoid substrate . The reaction proceeds through the formation of a key enzyme-acetyl intermediate at the A-cluster, which was confirmed through initial burst kinetic studies and direct chromatographic isolation of the active enzyme-acetyl species .

The process requires reductive activation of the A-cluster, which involves both electron transfer and protonation steps, highlighting the complex redox chemistry that underlies this catalytic function .

What electron transfer mechanisms occur during ACDS beta subunit catalysis?

The electron transfer mechanisms in the ACDS beta subunit involve complex redox transitions at the A-cluster. Research has demonstrated that the redox dependence of acetyl-CoA synthesis exhibits one-electron Nernst behavior, though this behavior actually represents the sum of two separate, low-potential, one-electron steps .

Titration experiments have established that two electrons are required for activation of the enzyme during the formation of the enzyme-acetyl intermediate. Subsequently, the A-cluster-acetyl species undergoes reductive elimination of the acetyl group, simultaneously releasing two low-potential electron equivalents .

This electron transfer mechanism is critical for understanding how the enzyme functions and has implications for:

• The redox state transitions of the Ni centers and Fe₄S₄ cluster

• The coupling of electron transfer to chemical bond formation and cleavage

• The potential role of electron trapping in facilitating the reaction under physiological redox conditions

Notably, researchers have found that trapping of electrons during the formation of the A-cluster-acetyl species from CO and methylcobinamide substrates is highly favorable, providing a means for extensive activation of the enzyme under otherwise nonpermissive physiological redox potentials .

What role does nickel play in the catalytic function of the ACDS beta subunit?

Nickel plays a crucial role in the catalytic function of the ACDS beta subunit as a key component of the A-cluster active site. The binuclear Ni-Ni center participates directly in the reaction chemistry of acetyl-CoA synthesis and cleavage .

Based on experimental evidence, the reaction mechanism appears to favor involvement of a [Fe₄S₄]¹⁺-Ni¹⁺ species over a [Fe₄S₄]²⁺-Ni⁰ form . This distinction is important for understanding the precise electronic states that facilitate catalysis. The nickel centers likely participate in:

• Binding of substrate molecules

• Electron transfer processes during catalysis

• Formation of organometallic intermediates with carbon-containing substrates

• Coordination chemistry changes during the catalytic cycle

Research has discussed the potential formation of a Ni²⁺-hydride intermediate, which may be facilitated by proton uptake during the reductive activation process . This suggests a complex role for the nickel centers beyond simple electron transfer, potentially including direct participation in hydrogen atom management during the reaction.

How do pH and redox potential affect the activity of recombinant ACDS beta subunit?

The activity of recombinant ACDS beta subunit is significantly influenced by both pH and redox potential, reflecting the complex electrochemical nature of its catalytic mechanism .

Research has demonstrated that the redox dependence of acetyl-CoA synthesis exhibits one-electron Nernst behavior. The effects of pH on the observed midpoint potential indicate that reductive activation of the A-cluster also involves protonation, highlighting the coupled nature of electron and proton transfer in this system .

The relationship between pH, redox potential, and enzyme activity can be summarized as follows:

Experiments monitoring acetyl-CoA formation at different pH values and redox potentials revealed initial burst kinetics under various conditions, indicating the formation of stoichiometric amounts of an A-cluster-acetyl adduct regardless of specific pH/redox conditions, though the rates and extents of reaction were affected .

What are the proposed mechanisms for acetyl-CoA synthesis at the A-cluster of ACDS beta subunit?

The mechanism of acetyl-CoA synthesis at the A-cluster involves several discrete steps and intermediate species. Based on experimental evidence, researchers have excluded mechanisms involving either one- or three-electron reduced forms of the A-cluster as immediate precursors to the acetyl species .

The current data favors a mechanism involving a [Fe₄S₄]¹⁺-Ni¹⁺ species over a [Fe₄S₄]²⁺-Ni⁰ form . The key steps in the proposed mechanism include:

• Reductive activation of the A-cluster requiring two electrons and protonation

• Binding of the methyl group from methylcobinamide (or methylated corrinoid protein)

• Coordination of CO at the active site

• Formation of an enzyme-acetyl intermediate (directly observed in experiments)

• CoA binding and transfer of the acetyl group to form acetyl-CoA

• Release of two low-potential electron equivalents during reductive elimination

The role of proton uptake in the possible formation of a Ni²⁺-hydride intermediate remains an area of active investigation and could represent a key step in the carbon-carbon bond formation process .

This complex mechanism integrates organometallic chemistry principles with biological electron transfer processes, making it a fascinating subject for both inorganic chemists and biochemists studying carbon fixation and energy metabolism.

How can researchers optimize the expression and purification of recombinant ACDS beta subunit?

Optimizing the expression and purification of recombinant ACDS beta subunit requires careful attention to several factors due to its complex metallocofactor and sensitivity to oxygen. Based on successful experimental approaches, researchers should consider:

Expression System Selection:

• Anaerobic expression systems are preferred due to the oxygen sensitivity of the iron-sulfur centers

• E. coli strains engineered for enhanced metalloproteins expression (e.g., with additional copies of rare tRNAs) may improve yields

• Expression under control of inducible promoters allows for optimization of induction timing and concentration

Culture Conditions:

• Growth media supplementation with nickel and iron sources to ensure proper metal incorporation

• Controlled growth temperatures (typically lower than standard, e.g., 18-25°C) to allow proper protein folding

• Anaerobic induction and harvest procedures to protect the metal centers

Purification Considerations:

• All purification steps should be performed under strict anaerobic conditions

• Buffer composition should include reducing agents (e.g., dithiothreitol or dithionite) to maintain reduced state

• Affinity chromatography with carefully chosen tags that don't interfere with metal center assembly

• Size exclusion and ion exchange chromatography for achieving high purity

Metallocofactor Reconstitution:

• In some cases, in vitro reconstitution of the A-cluster may be necessary if the heterologous expression system does not properly assemble the native cofactor

• Controlled addition of iron, sulfide, and nickel sources under reducing conditions

• Verification of metal content using spectroscopic methods such as EPR, XAS, or ICP-MS

Researchers should verify the activity of purified recombinant protein using activity assays such as acetyl-CoA synthesis with methylcobinamide as a substrate, which has been demonstrated to yield high rates of product formation with properly folded enzyme .

What spectroscopic methods are most effective for studying the A-cluster states in ACDS beta subunit?

Multiple spectroscopic techniques have proven valuable for characterizing the different redox and catalytic states of the A-cluster in the ACDS beta subunit. Each technique provides complementary information about the electronic and structural properties of this complex metallocofactor:

Electron Paramagnetic Resonance (EPR) Spectroscopy:

• Particularly useful for detecting paramagnetic states of the A-cluster

• Can distinguish between different redox states of the [Fe₄S₄] cluster

• Provides information about the electronic environment of Ni centers

• Low-temperature measurements can capture transient intermediates

X-ray Absorption Spectroscopy (XAS):

• Provides element-specific information about metal oxidation states and coordination environments

• XANES region gives oxidation state information for Ni and Fe centers

• EXAFS region provides metal-ligand distances and coordination numbers

• Particularly valuable for characterizing the Ni-Ni binuclear site

Mössbauer Spectroscopy:

• Offers detailed information about Fe oxidation states and electronic environments

• Can distinguish between different types of Fe sites within the [Fe₄S₄] cluster

• Valuable for tracking changes in the iron-sulfur cluster during catalysis

Infrared (IR) and Resonance Raman Spectroscopy:

• Useful for studying bound CO and other substrates at the active site

• Can provide information about metal-carbon bonds in organometallic intermediates

• Helps characterize the enzyme-acetyl intermediate species

UV-Visible Absorption Spectroscopy:

Researchers studying the ACDS beta subunit should consider using multiple spectroscopic approaches in combination to build a comprehensive understanding of the A-cluster states and transitions during catalysis .

How can researchers effectively study the kinetics of acetyl-CoA synthesis by recombinant ACDS beta subunit?

Studying the kinetics of acetyl-CoA synthesis by recombinant ACDS beta subunit requires specialized techniques due to the complex nature of the reaction and the oxygen sensitivity of the enzyme. Researchers can employ several methodological approaches:

Initial Burst Kinetics Analysis:

• This approach has successfully revealed the formation of stoichiometric amounts of an A-cluster-acetyl adduct

• Reactions are initiated under controlled redox conditions and quenched at specific time points

• Acetyl-CoA formation is quantified, typically using radioactive tracers or HPLC analysis

• The initial burst followed by steady-state rate provides evidence for rate-limiting steps in the catalytic cycle

Redox Titration Experiments:

• Control the redox potential using mediator dyes (like methyl viologen and benzyl viologen)

• Gradually adjust redox conditions using titanium(III) citrate or other reductants

• Monitor activity across a range of potentials to establish redox dependence

• Fit data to the Nernst equation to determine midpoint potentials

pH-Dependence Studies:

• Conduct activity assays across a range of pH values under controlled redox conditions

• Analyze the effect of pH on both midpoint potentials and catalytic rates

• Determine pKa values of important ionizable groups involved in catalysis

• Examine the coupling between electron transfer and protonation events

Substrate Binding and Product Formation Kinetics:

• Pre-steady-state kinetics using rapid mixing techniques (stopped-flow or quench-flow)

• Vary substrate concentrations to determine binding constants and rate-limiting steps

• Isotope labeling to track carbon and hydrogen transfer during the reaction

• Spectroscopic monitoring of reaction intermediates when possible

These methodological approaches have provided key insights into the mechanism of acetyl-CoA synthesis, revealing the two-electron requirement for enzyme activation, the formation of an enzyme-acetyl intermediate, and the reductive elimination process that releases two low-potential electron equivalents .

What approaches can be used to investigate the electron transfer pathways in ACDS beta subunit?

Investigating electron transfer pathways in the ACDS beta subunit requires specialized techniques that can probe the redox properties and electron movement within the complex metallocofactor. Researchers can employ several methodological approaches:

Protein Film Electrochemistry:

• Immobilize the enzyme on an electrode surface

• Directly measure electron transfer between the electrode and protein

• Determine formal potentials of different redox centers

• Study the kinetics of electron transfer under various conditions

Site-Directed Mutagenesis:

• Systematically modify residues potentially involved in electron transfer pathways

• Target conserved cysteine residues that coordinate the metal centers

• Evaluate the impact on redox properties and catalytic activity

• Identify key amino acids that facilitate electron movement between the [Fe₄S₄] cluster and Ni centers

Pulse Radiolysis and Flash Photolysis:

• Generate reducing equivalents rapidly and synchronously

• Monitor the kinetics of electron transfer through the protein using time-resolved spectroscopy

• Distinguish between different electron transfer steps based on their rates

• Determine activation parameters for electron transfer reactions

Computational Methods:

• Quantum mechanics/molecular mechanics (QM/MM) calculations

• Density functional theory (DFT) to model the electronic structure of the A-cluster

• Molecular dynamics simulations to identify potential electron transfer pathways

• Calculation of electronic coupling between redox centers

Spectroelectrochemical Analysis:

• Combine electrochemical control with spectroscopic observation

• Monitor spectral changes associated with different redox states

• Determine midpoint potentials for specific transitions

• Characterize intermediate species formed during electron transfer

Through these approaches, researchers have established that the redox dependence of acetyl-CoA synthesis follows one-electron Nernst behavior, representing the combined effect of two separate one-electron steps, and have identified the likely involvement of a [Fe₄S₄]¹⁺-Ni¹⁺ species in the catalytic mechanism .

How can researchers study the interaction between methylcobinamide and the ACDS beta subunit?

Studying the interaction between methylcobinamide (a mimic of the physiological base-off corrinoid substrate) and the ACDS beta subunit requires specialized techniques to capture this critical step in acetyl-CoA synthesis. Researchers can utilize several methodological approaches:

Binding Affinity Determination:

• Isothermal titration calorimetry (ITC) to measure thermodynamic parameters of binding

• Surface plasmon resonance (SPR) for real-time binding kinetics

• Fluorescence quenching if appropriate fluorophores can be introduced without disrupting function

• Equilibrium dialysis combined with analytical detection of methylcobinamide

Spectroscopic Characterization of the Bound Complex:

• UV-visible spectroscopy to monitor changes in the corrinoid absorption spectrum upon binding

• EPR spectroscopy to detect changes in the paramagnetic properties of both the corrinoid and A-cluster

• NMR spectroscopy with isotopically labeled methylcobinamide to map the binding interface

• Resonance Raman to probe the cobalt-carbon bond of methylcobinamide before and during catalysis

Kinetic Analysis of Methyl Transfer:

• Pre-steady-state kinetics to capture the initial methyl transfer event

• Quench-flow techniques combined with product analysis

• Isotope labeling (e.g., ¹³C-methyl group) to track the methyl transfer process

• Comparison of different methylcobinamide analogs to establish structure-activity relationships

Structural Studies:

• X-ray crystallography of the enzyme-substrate complex if crystals can be obtained

• Cryo-electron microscopy (cryo-EM) for structural determination without crystallization

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions of conformational change upon binding

• Computational docking and molecular dynamics simulations to predict binding modes

What are the major challenges in studying the complete reaction mechanism of ACDS beta subunit?

Investigating the complete reaction mechanism of the ACDS beta subunit presents several significant challenges that researchers must address through innovative approaches:

Capturing Transient Intermediates:

• The catalytic cycle likely involves short-lived organometallic species

• Requires rapid-freeze quenching techniques coupled with spectroscopy

• May need specially designed substrate analogs that slow particular steps

• Potentially requires time-resolved crystallography or spectroscopy

Distinguishing Between Proposed Mechanistic Models:

• Current evidence suggests a [Fe₄S₄]¹⁺-Ni¹⁺ species over a [Fe₄S₄]²⁺-Ni⁰ form

• Need for techniques that can definitively assign metal oxidation states

• Requirement for computational models that accurately represent the complex electronic structure

• Design of experiments that can distinguish between competing mechanistic hypotheses

Identifying Proton Transfer Pathways:

• Reductive activation involves coupled electron and proton transfer

• Need to identify specific residues involved in proton donation/acceptance

• Requires careful pH-dependent studies and isotope effect measurements

• May need to employ neutron diffraction for direct visualization of protons

Integrating Individual Steps into a Coherent Mechanism:

• The reaction involves multiple substrates entering in a coordinated sequence

• Challenge of determining the precise ordering and timing of substrate binding events

• Need to understand how the protein structure facilitates substrate channeling

• Requirement for techniques that can monitor multiple reaction parameters simultaneously

Addressing these challenges will require interdisciplinary approaches combining advanced spectroscopy, protein engineering, computational modeling, and the development of new methodological tools specifically designed for studying complex metalloenzymes .

How might the understanding of ACDS beta subunit inform bioinspired catalysts for carbon fixation?

The detailed understanding of ACDS beta subunit's structure and mechanism provides valuable insights for designing bioinspired catalysts for carbon fixation, which has significant implications for addressing climate change and developing sustainable chemistry:

Structural Features for Synthetic Catalyst Design:

• The binuclear Ni-Ni site bridged to an Fe₄S₄ cluster provides a template for synthetic metallocomplexes

• Understanding the specific coordination environment of the metals helps optimize ligand selection

• The spatial arrangement that facilitates C-C bond formation offers design principles for catalyst geometry

• Identification of key functional groups involved in substrate binding informs synthetic catalyst functionalization

Mechanistic Principles to Incorporate:

• The two-electron activation process suggests design parameters for redox-active catalysts

• Coupled electron/proton transfer highlights the importance of proton relay systems in synthetic designs

• The formation of metal-carbon bonds as key intermediates informs selection of metal centers

• Electron trapping mechanisms might be mimicked to enable catalysis under mild conditions

Practical Applications for Biomimetic Catalysts:

• Conversion of CO₂ to value-added carbon compounds

• Development of artificial carbon fixation systems for carbon capture technologies

• Creation of synthetic pathways for acetyl-CoA or similar two-carbon building blocks

• Integration into artificial photosynthetic systems for solar fuels production

Learning from Evolutionary Optimization:

• The A-cluster represents a highly evolved solution for carbon-carbon bond formation

• Understanding how the protein environment tunes metal reactivity informs catalyst support design

• Identifying conserved features across different organisms highlights essential catalytic elements

• Examining protein dynamics during catalysis informs design of flexible synthetic systems

By translating the fundamental insights from ACDS beta subunit research into synthetic catalysts, chemists can work toward more efficient and sustainable methods for carbon fixation that operate under mild conditions with earth-abundant metals .

What emerging technologies might advance our understanding of ACDS beta subunit function?

Several emerging technologies and methodological advances have the potential to significantly enhance our understanding of ACDS beta subunit function in the coming years:

Advanced Cryo-Electron Microscopy (Cryo-EM) Techniques:

• Time-resolved cryo-EM to capture intermediates during catalysis

• Improved resolution approaching atomic details for metallocofactors

• Ability to study the enzyme under more physiologically relevant conditions

• Visualization of conformational dynamics during substrate binding and product release

Quantum Biology Approaches:

• Application of quantum mechanical principles to understand electron tunneling pathways

• Investigation of quantum coherence effects in electron transfer processes

• Quantum entanglement studies between paired electrons in the A-cluster

• Development of quantum sensors for probing electronic states at unprecedented resolution

Artificial Intelligence and Machine Learning:

• Deep learning algorithms to predict reaction mechanisms from experimental data

• Neural networks trained on spectroscopic data to identify subtle patterns in intermediate states

• AI-assisted design of mutagenesis strategies to test mechanistic hypotheses

• Machine learning approaches to integrate diverse experimental datasets into coherent models

Novel Spectroscopic Techniques:

• Ultrafast two-dimensional infrared spectroscopy for tracking vibrational dynamics

• Femtosecond X-ray spectroscopy at free electron laser facilities

• Combined electron paramagnetic resonance and optical techniques for correlating electronic and structural changes

• Innovative spin labeling approaches to monitor conformational changes during catalysis

Single-Molecule Techniques:

• Single-enzyme activity measurements to capture heterogeneity in catalytic behavior

• Force spectroscopy to examine mechanical aspects of enzyme function

• Single-molecule FRET to monitor conformational dynamics during catalysis

• Nanoscale electrochemistry to study electron transfer at the individual enzyme level

These emerging technologies promise to provide unprecedented insights into the detailed mechanism of ACDS beta subunit function, potentially resolving current mechanistic ambiguities and opening new avenues for bioinspired catalyst design .