The role of water
in biological proton-coupled electron transfer
(PCET) is emerging as a key for understanding mechanistic details
at atomic resolution. Here we demonstrate
17
O high-frequency
electron–nuclear double resonance (ENDOR) in conjunction with
H
2
17
O-labeled protein buffer to establish the
presence of ordered water molecules at three radical intermediates
in an active enzyme complex, the α
2
β
2
E. coli
ribonucleotide reductase.
Our data give unambiguous evidence that all three, individually trapped,
intermediates are hyperfine coupled to one water molecule with Tyr-O···
17
O distances in the range 2.8–3.1 Å. The availability
of this structural information will allow for quantitative models
of PCET in this prototype enzyme. The results also provide a spectroscopic
signature for water H-bonded to a tyrosyl radical.
Ribonucleotide reductases
(RNRs) catalyze the reduction of ribonucleotides
to deoxyribonucleotides, thereby playing a key role in DNA replication
and repair. Escherichia coli class
Ia RNR is an α2β2 enzyme complex
that uses a reversible multistep radical transfer (RT) over 32 Å
across its two subunits, α and β, to initiate, using its
metallo-cofactor in β2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled
electron-transfer (PCET) process. An unresolved step is the RT involving
Y356(β) and Y731(α) across the α/β
interface. Using 2,3,5-F3Y122-β2 with 3,5-F2Y731‑α2, GDP (substrate) and TTP (allosteric effector), a Y356
• intermediate was trapped and its identity was
verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz
pulse electron–electron double resonance spectroscopies. 94
GHz 19F electron-nuclear double resonance spectroscopy
allowed measuring the interspin distances between Y356
• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the
double mutant E52Q/F3Y122-β2 were carried out for comparison to the recently published
cryo-EM structure of a holo RNR complex. For both mutant combinations,
the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with
3,5-F2Y731 within the H-bond distance to Y356
•, whereas the second one is consistent
with the conformation observed in the cryo-EM structure. The observations
unexpectedly suggest the possibility of a colinear PCET, in which
electron and proton are transferred from the same donor to the same
acceptor between Y356 and Y731. The results
highlight the important role of state-of-the-art EPR spectroscopy
to decipher this mechanism.
A systematic review and analysis of the most stable spatial arrangements of n carbon, n oxygen, and 2n hydrogen atoms including vibrational zero-point energy up to n = 5 shows that small-molecule aggregates win, typically followed by thermally unstable molecules, before kinetically stable molecules and finally carbohydrates are found. Near n ≈ 60 a crossover to carbon allotropes and ice as the global minimum structure is expected and the asymptotic limit is most likely graphite and ice. Implications for astrochemical and fermentation processes are discussed. Density functionals like B3LYPD3 are found to describe these energy sequences quite poorly, mostly due to an overestimated stability of carbon in high oxidation states.
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