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Nitrogenases are a class of enzymes produced by certain bacteria, such as cyanobacteria and rhizobacteria, that catalyze the reduction of atmospheric nitrogen to ammonia.
Answer: True
Explanation: Nitrogenases are enzymes synthesized by specific bacteria, including cyanobacteria and rhizobacteria, which are responsible for catalyzing the reduction of atmospheric nitrogen (N₂) into ammonia (NH₃).
Nitrogen fixation is a vital process for all forms of life because nitrogen is an essential element for the biosynthesis of nucleotides and amino acids.
Answer: True
Explanation: Nitrogen fixation is indispensable for all life forms, as nitrogen is a fundamental component required for the biosynthesis of critical biological macromolecules like nucleotides and amino acids.
Nitrogenase acts as a catalyst to significantly reduce the high activation energy required for the thermodynamically favorable formation of ammonia from nitrogen and hydrogen.
Answer: True
Explanation: Nitrogenase functions as a catalyst, effectively lowering the substantial activation energy barrier for the synthesis of ammonia from nitrogen and hydrogen, a reaction that is thermodynamically favorable but kinetically slow.
*Azospirillum* and *Frankia* are examples of free-living bacteria that synthesize nitrogenase.
Answer: False
Explanation: *Azospirillum* and *Frankia* are examples of *mutualistic, symbiotic* bacteria that synthesize nitrogenase, not free-living ones. Free-living examples include cyanobacteria and *Azotobacter*.
*Rhizobium* is a mutualistic bacterium that synthesizes nitrogenase and forms symbiotic relationships with legumes.
Answer: True
Explanation: *Rhizobium* is a well-known mutualistic bacterium that produces nitrogenase and establishes symbiotic associations with leguminous plants, facilitating nitrogen fixation.
What is the primary biological function of nitrogenases?
Answer: To catalyze the reduction of atmospheric nitrogen (N₂) to ammonia (NH₃).
Explanation: The fundamental biological role of nitrogenases is to catalyze the reduction of atmospheric dinitrogen (N₂) into ammonia (NH₃), making nitrogen available for biological processes.
Why is nitrogen fixation considered a vital process for all forms of life?
Answer: It is essential for the biosynthesis of fundamental biological molecules like nucleotides and amino acids.
Explanation: Nitrogen fixation is crucial because it converts atmospheric nitrogen into a usable form, which is then incorporated into essential biological molecules such as nucleotides and amino acids, vital for all life.
How do nitrogenases facilitate the formation of ammonia from nitrogen and hydrogen?
Answer: By acting as a catalyst to significantly reduce the high activation energy barrier.
Explanation: Nitrogenases function as catalysts, effectively lowering the substantial activation energy barrier for the thermodynamically favorable, but kinetically slow, reaction of ammonia formation from nitrogen and hydrogen.
Which of the following is an example of a free-living bacterium that synthesizes nitrogenase?
Answer: Azotobacter
Explanation: *Azotobacter* is an example of a free-living, non-symbiotic bacterium capable of synthesizing nitrogenase for atmospheric nitrogen fixation.
Which mutualistic bacterium is associated with legumes and synthesizes nitrogenase?
Answer: Rhizobium
Explanation: *Rhizobium* is a mutualistic bacterium that forms symbiotic relationships with legumes, residing in root nodules where it synthesizes nitrogenase to fix atmospheric nitrogen.
The Fe-only protein component of nitrogenase is a heterotetramer, while the MoFe protein is a homodimer.
Answer: False
Explanation: The Fe-only protein (reductase) is a homodimer, whereas the MoFe protein is a heterotetramer, composed of two alpha and two beta subunits.
The FeMo cofactor is a crucial component located within the alpha subunits of the MoFe protein, serving as the active site for nitrogen fixation.
Answer: True
Explanation: The FeMo cofactor is indeed a vital part of the nitrogenase enzyme, situated within the alpha subunits of the MoFe protein, and functions as the active site where atmospheric nitrogen is reduced to ammonia.
The reductase (Fe protein) is a monomeric protein containing two [Fe₄S₄] clusters and directly reduces N₂ to NH₃.
Answer: False
Explanation: The reductase (Fe protein) is a dimer composed of two identical subunits, containing one [Fe₄S₄] cluster, and its role is to transfer electrons from a reducing agent to the MoFe protein, not to directly reduce N₂ to NH₃.
The MoFe protein is a homodimer with a mass of approximately 60-64 kDa, containing only P-clusters.
Answer: False
Explanation: The MoFe protein is a heterotetramer with an approximate mass of 240-250 kDa, and it contains both P-clusters and FeMo cofactors.
Recent evidence suggests that the oxidation state of Molybdenum (Mo) in MoFe nitrogenases is Mo(III), rather than the previously believed Mo(V).
Answer: True
Explanation: Recent research indicates that the oxidation state of molybdenum within the MoFe nitrogenase is Mo(III), a revision from the earlier understanding of Mo(V).
The P-cluster within the MoFe protein has a core structure of (Fe₆S₆) and is linked to the protein by four histidine residues.
Answer: False
Explanation: The P-cluster in the MoFe protein has a core structure of (Fe₈S₇) and is linked to the protein by six cysteine residues, not four histidine residues.
Each FeMo cofactor has the chemical formula (Fe₇MoS₉C) and is covalently attached to the alpha subunit through one cysteine and one histidine residue.
Answer: True
Explanation: The FeMo cofactor is accurately described by the chemical formula (Fe₇MoS₉C) and is covalently linked to the alpha subunit of the MoFe protein via one cysteine and one histidine residue.
Which two main components typically form a nitrogenase assembly?
Answer: The homodimeric Fe-only protein and the heterotetrameric MoFe protein.
Explanation: A typical nitrogenase enzyme complex is composed of two primary components: the homodimeric Fe-only protein (reductase) and the heterotetrameric MoFe protein.
What is the primary role of the FeMo cofactor in the nitrogenase enzyme?
Answer: To serve as the active site where nitrogen fixation occurs, binding the N₂ molecule.
Explanation: The FeMo cofactor functions as the active site within the MoFe protein where the dinitrogen molecule binds and is subsequently reduced to ammonia.
Which of the following accurately describes the reductase (Fe protein) component of nitrogenase?
Answer: It is a dimer composed of two identical subunits, containing one [Fe₄S₄] cluster.
Explanation: The reductase, or Fe protein, is a homodimer consisting of two identical subunits, each containing a single [Fe₄S₄] cluster, and its role is to transfer electrons from a reducing agent to the MoFe protein.
Which of the following accurately describes the structural characteristics of the MoFe protein?
Answer: It is a heterotetramer composed of two alpha and two beta subunits, with a mass of 240-250 kDa.
Explanation: The MoFe protein is a heterotetramer, comprising two alpha and two beta subunits, and has an approximate mass of 240-250 kDa.
What is the currently suggested oxidation state of Molybdenum (Mo) in MoFe nitrogenases?
Answer: Mo(III)
Explanation: Current evidence indicates that the oxidation state of molybdenum in MoFe nitrogenases is Mo(III), a revision from earlier assumptions.
What is the core structure of the P-cluster within the MoFe protein?
Answer: (Fe₈S₇)
Explanation: The P-cluster within the MoFe protein possesses a core structure of (Fe₈S₇), formed by two interconnected [Fe₄S₃] cubes.
How is the FeMo cofactor covalently attached to the alpha subunit of the MoFe protein?
Answer: Through one cysteine residue and one histidine residue.
Explanation: The FeMo cofactor is covalently linked to the alpha subunit of the MoFe protein via a cysteine residue and a histidine residue.
The transfer of electrons by the Fe protein is powered by the binding and subsequent hydrolysis of adenosine triphosphate (ATP).
Answer: True
Explanation: The energy required for electron transfer by the Fe protein is supplied through the binding and subsequent hydrolysis of ATP, which also induces conformational changes necessary for the interaction between the Fe and MoFe proteins.
Electrons flow directly from the Fe protein to the FeMo cofactors, bypassing the P-clusters entirely.
Answer: False
Explanation: Electrons from the Fe protein first enter the MoFe protein at the P-clusters, and then are transferred from the P-clusters to the FeMo cofactors, meaning the P-clusters are not bypassed.
The Lowe-Thorneley kinetic model describes the sequential addition of eight proton and electron equivalents during the reduction of nitrogen to ammonia.
Answer: True
Explanation: The Lowe-Thorneley kinetic model precisely outlines the sequential transfer of eight proton and electron equivalents to the FeMo-cofactor, leading to the reduction of nitrogen to ammonia.
The Lowe-Thorneley model requires 6 equivalents of protons and electrons for nitrogen reduction, which is fewer than predicted by the balanced chemical reaction.
Answer: False
Explanation: The Lowe-Thorneley model specifies that 8 equivalents of protons and electrons are required for nitrogen reduction, which is *more* than the 6 equivalents predicted by the balanced chemical reaction.
The E₀ intermediate in the Lowe-Thorneley model represents the resting state of the nitrogenase enzyme before the catalytic process begins.
Answer: True
Explanation: The E₀ intermediate is defined as the inactive, resting state of the nitrogenase enzyme prior to the initiation of the nitrogen fixation catalytic cycle.
The E₂ intermediate is proposed to contain the metal cluster in an oxidized state with two free protons.
Answer: False
Explanation: The E₂ intermediate is proposed to contain the metal cluster in its *resting oxidation state*, with two added electrons stored as a bridging hydride and a proton bonded to a sulfur atom.
The E₄ intermediate, known as the Janus intermediate, is significant because it can either revert to the E₀ resting state or proceed with nitrogen binding.
Answer: True
Explanation: The E₄ intermediate, or Janus intermediate, is a crucial branching point in the catalytic cycle, capable of either returning to the E₀ resting state or advancing to bind nitrogen and continue fixation.
The E₄ intermediate was first observed in a wild-type enzyme using X-ray crystallography, showing a single bridging hydride.
Answer: False
Explanation: The E₄ intermediate was first observed using freeze-quench techniques with a *mutated* protein, not a wild-type enzyme, and ENDOR experiments revealed the presence of *two* bridging hydrides.
The 'distal' and 'alternating' pathways are the two main hypotheses for the mechanism of N₂ fixation after the E₄ complex, differing in hydrogen addition sequence.
Answer: True
Explanation: Following the E₄ intermediate, the 'distal' and 'alternating' pathways represent the primary mechanistic hypotheses for N₂ reduction, distinguished by their distinct sequences of hydrogen addition to the dinitrogen molecule.
In the distal pathway, hydrogen additions alternate between the terminal and metal-bound nitrogen atoms until ammonia is released.
Answer: False
Explanation: In the distal pathway, the terminal nitrogen atom is hydrogenated first, leading to ammonia release, before the metal-bound nitrogen is hydrogenated. The alternating pathway involves alternating hydrogen additions.
MgATP binding and its subsequent hydrolysis provide the necessary energy for electron transfer from the Fe protein to the MoFe protein.
Answer: True
Explanation: The energy required for the crucial electron transfer from the Fe protein to the MoFe protein is supplied by the binding and subsequent hydrolysis of MgATP.
Upon MgATP binding to the Fe protein, a salt bridge forms between Lysine 15 and Aspartic acid 125, stabilizing the protein.
Answer: False
Explanation: In the absence of MgATP, a salt bridge exists between Lysine 15 and Aspartic acid 125; this salt bridge is *interrupted* upon MgATP binding, not formed.
MgATP binding induces a conformational change in the Fe protein, causing the entire protein to contract and decreasing its radius by approximately 2.0 Å.
Answer: True
Explanation: Binding of MgATP to the Fe protein triggers a significant conformational change, resulting in the contraction of the entire protein and a reduction in its radius by approximately 2.0 Å.
What powers the transfer of electrons by the Fe protein in the nitrogenase complex?
Answer: The binding and hydrolysis of adenosine triphosphate (ATP).
Explanation: The energy required for the Fe protein to transfer electrons is supplied by the binding and subsequent hydrolysis of ATP.
What is the correct path of electrons through the nitrogenase complex?
Answer: Fe protein -> P-clusters -> FeMo cofactors.
Explanation: Electrons are transferred sequentially from the Fe protein to the P-clusters within the MoFe protein, and then finally to the FeMo cofactors, where nitrogen reduction occurs.
What does the Lowe-Thorneley kinetic model describe in the context of nitrogen fixation?
Answer: The sequential addition of proton and electron equivalents to the FeMo-cofactor during nitrogen reduction.
Explanation: The Lowe-Thorneley kinetic model elucidates the step-by-step addition of proton and electron equivalents to the FeMo-cofactor, which drives the reduction of nitrogen to ammonia.
According to the Lowe-Thorneley model, how many equivalents of protons and electrons are required for nitrogen reduction, and how does this compare to the balanced chemical reaction?
Answer: 8 equivalents, which is more than predicted by the balanced chemical reaction.
Explanation: The Lowe-Thorneley model postulates that nitrogen reduction necessitates 8 equivalents of protons and electrons, a quantity exceeding the 6 equivalents predicted by the stoichiometric balanced chemical reaction.
What characterizes the E₀ intermediate in the Lowe-Thorneley kinetic model?
Answer: It represents the resting state of the nitrogenase enzyme before catalysis begins.
Explanation: The E₀ intermediate signifies the quiescent, resting state of the nitrogenase enzyme prior to the initiation of its catalytic activity in nitrogen fixation.
What is the proposed composition of the E₂ intermediate in the Lowe-Thorneley model?
Answer: The metal cluster in its resting oxidation state, with two added electrons in a bridging hydride and a proton bonded to sulfur.
Explanation: The E₂ intermediate is hypothesized to consist of the metal cluster in its resting oxidation state, incorporating two electrons within a bridging hydride and an additional proton bound to a sulfur atom.
Why is the E₄ intermediate, also known as the Janus intermediate, significant in the Lowe-Thorneley model?
Answer: It occurs after half of the electron and proton transfers and can either revert to E₀ or proceed with nitrogen binding.
Explanation: The E₄ intermediate, or Janus intermediate, is pivotal as it marks the halfway point of electron and proton transfers, offering a bifurcation where the enzyme can either return to its resting state (E₀) or commit to nitrogen binding and further reduction.
How was the E₄ intermediate first observed and characterized?
Answer: Using freeze-quench techniques with a mutated protein (Valine 70 to Isoleucine) that prevented substrate access.
Explanation: The E₄ intermediate was initially detected and characterized using freeze-quench techniques on a mutated nitrogenase protein (Valine 70 to Isoleucine) engineered to restrict substrate access to the FeMo-cofactor.
What are the two main hypotheses for the mechanism of N₂ fixation after the E₄ complex?
Answer: The distal pathway and the alternating pathway.
Explanation: Following the E₄ complex, the two predominant mechanistic hypotheses for N₂ fixation are the 'distal' pathway and the 'alternating' pathway, which describe different sequences of hydrogen addition.
How does the 'distal' pathway for N₂ fixation differ from the 'alternating' pathway?
Answer: The distal pathway hydrogenates the terminal nitrogen first, releases ammonia, then hydrogenates the metal-bound nitrogen.
Explanation: The distal pathway is characterized by the initial hydrogenation of the terminal nitrogen atom, followed by ammonia release, and then subsequent hydrogenation of the metal-bound nitrogen, contrasting with the alternating pathway's sequential additions.
What is the primary role of MgATP binding in the nitrogenase mechanism?
Answer: To provide the necessary energy for electron transfer from the Fe protein to the MoFe protein.
Explanation: MgATP binding and its hydrolysis are crucial for supplying the energetic input required to facilitate electron transfer from the Fe protein to the MoFe protein, a key step in nitrogen fixation.
Which amino acid residues are primarily involved in MgATP binding to the Fe protein, and what happens to the salt bridge between Lysine 15 and Aspartic acid 125 upon binding?
Answer: Lysine 15, Aspartic acid 125, and Serine 16; the salt bridge is interrupted.
Explanation: Lysine 15, Aspartic acid 125, and Serine 16 are key residues in MgATP binding to the Fe protein, and this binding event leads to the interruption of the salt bridge between Lysine 15 and Aspartic acid 125.
What conformational change does MgATP binding induce in the Fe protein?
Answer: It causes the entire protein to contract, decreasing its radius by approximately 2.0 Å.
Explanation: The binding of MgATP to the Fe protein triggers a conformational shift that causes the entire protein to contract, resulting in an approximate 2.0 Å decrease in its radius.
Alternative nitrogenases become active in environments where there is a low availability of the molybdenum (Mo) cofactor.
Answer: True
Explanation: Alternative nitrogenases are expressed and become active under conditions where molybdenum, a key component of the standard MoFe nitrogenase, is scarce.
The two known types of alternative nitrogenases are the nickel-iron (NiFe) type and the copper-iron (CuFe) type.
Answer: False
Explanation: The two recognized types of alternative nitrogenases are the vanadium-iron (VFe) type and the iron-iron (FeFe) type, not nickel-iron or copper-iron.
Alternative nitrogenases share homologous alpha and beta subunits with the MoFe nitrogenase, indicating a shared evolutionary origin.
Answer: True
Explanation: Alternative nitrogenases possess alpha and beta subunits that are homologous to those found in the MoFe nitrogenase, suggesting a common evolutionary lineage.
All nitrogenases, including alternative types, utilize molybdenum (Mo) as the central metal in their cofactors.
Answer: False
Explanation: While standard nitrogenase uses molybdenum, alternative nitrogenases utilize vanadium or only iron as the central metal in their cofactors, not molybdenum.
In the FeFe nitrogenase, the δ/γ subunit plays a role in helping to bind the cofactor, assisting in adaptation to new metals.
Answer: True
Explanation: The δ/γ subunit in FeFe nitrogenase is involved in cofactor binding, a function believed to have facilitated the enzyme's adaptation to different metal cofactors during evolution.
Organisms generally prefer alternative nitrogenases when molybdenum is available because they are more efficient in ATP usage than MoFe nitrogenase.
Answer: False
Explanation: Organisms prefer the MoFe nitrogenase when molybdenum is available because it is the most efficient type, consuming less ATP for proton reduction compared to alternative nitrogenases.
Vanadium nitrogenases uniquely catalyze the conversion of carbon monoxide into alkanes, a reaction comparable to Fischer-Tropsch synthesis.
Answer: True
Explanation: Vanadium nitrogenases exhibit a unique catalytic capability, converting carbon monoxide into alkanes, a process mechanistically similar to the industrial Fischer-Tropsch synthesis.
Under what environmental conditions do alternative nitrogenases typically become active?
Answer: Low availability of the molybdenum (Mo) cofactor.
Explanation: Alternative nitrogenases are typically activated in environments characterized by a limited supply of molybdenum, which is essential for the standard MoFe nitrogenase.
What are the two known types of alternative nitrogenases?
Answer: Vanadium-iron (VFe) and Iron-iron (FeFe).
Explanation: The two identified types of alternative nitrogenases are the vanadium-iron (VFe) type and the iron-iron (FeFe) type.
How do the structures of alternative nitrogenases compare to the MoFe nitrogenase?
Answer: They consist of two alpha, two beta, and two delta (or gamma) subunits, with alpha and beta homologous to MoFe nitrogenase.
Explanation: Alternative nitrogenases are composed of two alpha, two beta, and two delta (or gamma) subunits, where the alpha and beta subunits share homology with those found in the MoFe nitrogenase.
What is the key difference in the metal cofactors between standard and alternative nitrogenases?
Answer: Standard nitrogenase uses molybdenum, while alternative types use vanadium or only iron.
Explanation: The primary distinction in metal cofactors is that standard nitrogenase incorporates molybdenum, whereas alternative nitrogenases utilize either vanadium or solely iron.
What is the primary role attributed to the δ/γ subunit in alternative nitrogenases, especially in the FeFe type?
Answer: Helping to bind the cofactor.
Explanation: The δ/γ subunit in alternative nitrogenases, particularly the FeFe type, is primarily involved in facilitating the binding of the cofactor, a role that likely contributed to the enzyme's adaptation to various metals.
Why is the MoFe nitrogenase generally preferred by organisms when molybdenum is available?
Answer: It is the most efficient type, wasting less ATP on proton reduction.
Explanation: The MoFe nitrogenase is favored when molybdenum is accessible because it demonstrates superior efficiency, minimizing ATP expenditure on the unproductive reduction of protons to dihydrogen compared to alternative nitrogenases.
What unique reaction can vanadium nitrogenases catalyze involving carbon monoxide?
Answer: The conversion of carbon monoxide into alkanes, similar to Fischer-Tropsch synthesis.
Explanation: Vanadium nitrogenases possess the distinct ability to catalyze the conversion of carbon monoxide into alkanes, a reaction mechanistically analogous to the Fischer-Tropsch synthesis.
Nitrogenases can reduce carbon dioxide to methane, a reaction that is highly specific to the enzyme.
Answer: False
Explanation: Nitrogenases can reduce carbon dioxide to carbon monoxide and water, but not methane. Furthermore, nitrogenases are known to reduce a variety of non-specific substrates, indicating a lack of high specificity for reactions other than N₂ reduction.
Dihydrogen (H₂) acts as a competitive inhibitor of nitrogenase, while carbon monoxide (CO) functions as a non-competitive inhibitor.
Answer: True
Explanation: Dihydrogen (H₂) competitively inhibits nitrogenase, whereas carbon monoxide (CO) acts as a non-competitive inhibitor by preventing the binding of dinitrogen to the active site.
Oxygen is generally beneficial for nitrogenase activity, as it helps to remove excess electrons from the active site.
Answer: False
Explanation: Oxygen is detrimental to most nitrogenases, causing irreversible inhibition by oxidatively degrading their iron-sulfur cofactors, and is not beneficial for enzyme activity.
Besides dinitrogen, which of the following is a non-specific substrate that nitrogenase can reduce?
Answer: Acetylene to ethylene.
Explanation: Beyond its primary function of reducing dinitrogen, nitrogenase can also non-specifically reduce other substrates, such as acetylene to ethylene.
Which molecule acts as a non-competitive inhibitor of nitrogenase by preventing dinitrogen binding?
Answer: Carbon monoxide (CO)
Explanation: Carbon monoxide (CO) functions as a non-competitive inhibitor of nitrogenase, impeding the enzyme's activity by preventing the binding of dinitrogen to its active site.
How do some nitrogen-fixing organisms, such as Azotobacteraceae, protect nitrogenase from oxygen?
Answer: By maintaining a high metabolic rate to reduce oxygen at the cell membrane.
Explanation: Certain nitrogen-fixing organisms, such as Azotobacteraceae, employ strategies like maintaining a high metabolic rate to rapidly consume oxygen at the cell membrane, thereby protecting the oxygen-sensitive nitrogenase enzyme.
Nitrogenases are exclusively encoded by a single, highly conserved gene across all nitrogen-fixing organisms.
Answer: False
Explanation: Nitrogenases are encoded by *Nif* genes or their homologous counterparts, which are not a single, highly conserved gene but rather a family of genes that can vary across different nitrogen-fixing organisms.
Nitrogenases are divided into two main groups, with alternative nitrogenases found in Class I.
Answer: False
Explanation: Nitrogenases are categorized into three main groups or classes (I, II, and III), with alternative nitrogenases specifically nested within Class III.
Nif-I nitrogenases are primarily found in aerobic or facultatively anaerobic diazotrophs that possess large *Nif* gene families.
Answer: True
Explanation: Nif-I nitrogenases are predominantly observed in aerobic or facultatively anaerobic diazotrophs, which are characterized by extensive *Nif* gene families.
Ancestral sequence reconstruction indicates that early Group I nitrogenases operated more rapidly and with higher ATP efficiency than modern versions.
Answer: False
Explanation: Ancestral sequence reconstruction studies suggest that early Group I nitrogenases operated more slowly than modern versions, though they maintained a similar efficiency in ATP usage.
The NifEN maturase has a structure quite similar to nitrogenase component I (NifDK) and is involved in assembling P-cluster and M-cluster precursors.
Answer: True
Explanation: The NifEN maturase exhibits structural homology to nitrogenase component I (NifDK) and plays a critical role in the assembly and transfer of P-cluster and M-cluster precursors.
The structural dissimilarity between NifEN and NifDK, along with NifEN's inability to fix nitrogen, supports their independent evolutionary origins.
Answer: False
Explanation: The structural *similarity* between NifEN and NifDK, coupled with NifEN's nitrogen-fixing ability when expressed in *E. coli*, supports the hypothesis of their evolution from a gene duplication event, not independent origins.
Isotopic data suggests that molybdenum-based nitrogen fixation is an ancient process, at least 3.2 gigayears old.
Answer: True
Explanation: Isotopic evidence indicates that molybdenum-based nitrogen fixation is a very ancient biological process, dating back at least 3.2 gigayears.
Nitrogenase subunits exhibit significant sequence similarity to subunits of the light-dependent version of protochlorophyllide reductase.
Answer: False
Explanation: Nitrogenase subunits show significant sequence similarity to the subunits of the *light-independent* version of protochlorophyllide reductase (DPOR), not the light-dependent version.
'Class IV' *nif* genes in methanogens, such as the CfbC type, are known to produce coenzyme F430, even though they do not fix nitrogen.
Answer: True
Explanation: 'Class IV' *nif* genes, found in methanogens, are homologs of nitrogenase subunits that do not fix nitrogen but are involved in other metabolic pathways, such as the CfbC type producing coenzyme F430.
Which genes are responsible for encoding nitrogenases?
Answer: Nif genes
Explanation: Nitrogenases are encoded by a specific set of genes known as *Nif* genes or their homologous counterparts.
Into how many main groups or clades are nitrogenases divided, and where are alternative nitrogenases found?
Answer: Three groups; Class III.
Explanation: Nitrogenases are classified into three primary groups (Classes I, II, and III), with the alternative nitrogenases specifically categorized within Class III.
What is the general distribution pattern of Nif-I nitrogenases?
Answer: Primarily found in aerobic or facultatively anaerobic diazotrophs with large *Nif* gene families.
Explanation: Nif-I nitrogenases are predominantly observed in aerobic or facultatively anaerobic diazotrophs, which are characterized by the presence of large *Nif* gene families.
What did ancestral sequence reconstruction reveal about early Group I nitrogenases like Anc2 and Anc1?
Answer: They operated more slowly than modern versions but maintained similar ATP usage efficiency.
Explanation: Ancestral sequence reconstruction studies indicated that early Group I nitrogenases, such as Anc2 and Anc1, functioned at a slower rate than their modern counterparts but retained comparable ATP usage efficiency.
What is the relationship between the NifEN maturase and nitrogenase component I (NifDK)?
Answer: NifEN is structurally similar to NifDK and is involved in assembling and transferring P-cluster and M-cluster precursors to NifDK.
Explanation: The NifEN maturase exhibits structural homology to nitrogenase component I (NifDK) and is functionally involved in the assembly and transfer of P-cluster and M-cluster precursors.
What evidence supports the hypothesis that NifEN and NifDK evolved from a gene duplication event?
Answer: The structural similarity between NifEN and NifDK, and NifEN's nitrogen-fixing ability in *E. coli*.
Explanation: The structural resemblance between NifEN and NifDK, coupled with NifEN's demonstrated nitrogen-fixing capability when expressed in *E. coli*, provides evidence for their evolutionary origin from a gene duplication event.
According to isotopic data, what is the approximate age of molybdenum-based nitrogen fixation?
Answer: At least 3.2 gigayears old.
Explanation: Isotopic analyses suggest that molybdenum-based nitrogen fixation is an ancient process, with an estimated age of at least 3.2 gigayears.
To which other enzyme do nitrogenase subunits show significant sequence similarity, placing them in the same superfamily?
Answer: Light-independent protochlorophyllide reductase (DPOR).
Explanation: Nitrogenase subunits exhibit substantial sequence similarity to the subunits of light-independent protochlorophyllide reductase (DPOR), indicating their membership in the same enzyme superfamily.
What is a known function of 'Class IV' *nif* genes in methanogens?
Answer: Producing coenzyme F430 (CfbC type) or synthesizing methionine, ethylene, and methane (Mar type).
Explanation: In methanogens, 'Class IV' *nif* genes, while not involved in nitrogen fixation, are known to perform other functions, such as the CfbC type producing coenzyme F430, or the Mar type synthesizing methionine, ethylene, and methane.
The acetylene reduction assay (ARA) measures nitrogenase activity by quantifying the conversion of ethylene gas to acetylene gas.
Answer: False
Explanation: The acetylene reduction assay (ARA) quantifies nitrogenase activity by measuring the conversion of *acetylene gas to ethylene gas*, not the reverse.
A limitation of the acetylene reduction assay (ARA) is that dihydrogen (H₂) competes with N₂ but not acetylene, which can lead to an overestimation of nitrogenase activity.
Answer: True
Explanation: A known limitation of the acetylene reduction assay (ARA) is that dihydrogen (H₂) competes with dinitrogen (N₂) for the enzyme's active site but does not compete with acetylene, potentially leading to an inflated estimation of nitrogenase activity.
What is a common laboratory method for estimating nitrogenase activity?
Answer: The acetylene reduction assay (ARA).
Explanation: The acetylene reduction assay (ARA) is a widely employed laboratory technique for quantifying nitrogenase activity, leveraging the enzyme's ability to reduce acetylene to ethylene.
Which of the following is a limitation of the acetylene reduction assay (ARA)?
Answer: It can overestimate nitrogenase activity because dihydrogen (H₂) competes with N₂ but not acetylene.
Explanation: A significant limitation of the acetylene reduction assay (ARA) is its potential to overestimate nitrogenase activity due to dihydrogen (H₂) competing with dinitrogen (N₂) for the enzyme, but not with acetylene.