Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis.

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. Borosin natural product pathways are the only known ribosomally encoded and posttranslationally modified peptides (RiPPs) pathways to incorporate backbone α-N-methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed 'split borosins'), featuring an iteratively acting α-N-methyltransferase and separate precursor peptide substrate from the metal-respiring bacterium Shewanella oneidensis.

Mechanism of N Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H.

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N reduction relative to H evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H is a key feature in the N reduction mechanism. The N reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown.

Mechanism of Nitrogenase H Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects.

Nitrogenase catalyzes the reduction of dinitrogen (N) to two ammonia (NH) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps.

Negative cooperativity in the nitrogenase Fe protein electron delivery cycle.

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N) to two ammonia (NH) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the αβ MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two P for each ET, P release, and dissociation of oxidized Fe protein-(ADP) from the MoFe protein.

Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium.

Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO2) to methane (CH4). Here, we developed the anoxygenic phototroph, Rhodopseudomonas palustris, as a biocatalyst capable of light-driven CO2 reduction to CH4 in vivo using this remodeled nitrogenase.

Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle.

Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state.

Fe protein-independent substrate reduction by nitrogenase MoFe protein variants.

The reduction of substrates catalyzed by nitrogenase normally requires nucleotide-dependent Fe protein delivery of electrons to the MoFe protein, which contains the active site FeMo cofactor. Here, it is reported that independent substitution of three amino acids (β-98(Tyr→His), α-64(Tyr→His), and β-99(Phe→His)) located between the P cluster and FeMo cofactor within the MoFe protein endows it with the ability to reduce protons to H2, azide to ammonia, and hydrazine to ammonia without the need for Fe protein or ATP. Instead, electrons can be provided by the low-potential reductant polyaminocarboxylate-ligated Eu(II) (Em values of -1.

Nitrite and hydroxylamine as nitrogenase substrates: mechanistic implications for the pathway of N₂ reduction.

Investigations of reduction of nitrite (NO2(-)) to ammonia (NH3) by nitrogenase indicate a limiting stoichiometry, NO2(-) + 6e(-) + 12ATP + 7H(+) → NH3 + 2H2O + 12ADP + 12Pi. Two intermediates freeze-trapped during NO2(-) turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N2 reduction. The proposed NO2(-) reduction intermediate hydroxylamine (NH2OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped.

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s(-1), 25 °C.