Vitamin B(12) (cyanocobalamin, CNCbl) and its derivatives are structurally complex and functionally diverse biomolecules. The excited state and radical pair reaction dynamics that follow their photoexcitation have been previously studied in detail using UV-visible techniques. Similar time-resolved infrared (TRIR) data are limited, however. Herein we present TRIR difference spectra in the 1300-1700 cm(-1) region between 2 ps and 2 ns for adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), CNCbl, and hydroxocobalamin (OHCbl). The spectral profiles of all four cobalamins are complex, with broad similarities that suggest the vibrational excited states are related, but with a number of identifiable variations. The majority of the signals from AdoCbl and MeCbl decay with kinetics similar to those reported in the literature from UV-visible studies. However, there are regions of rapid (<10 ps) vibrational relaxation (peak shifts to higher frequencies from 1551, 1442, and 1337 cm(-1)) that are more pronounced in AdoCbl than in MeCbl. The AdoCbl data also exhibit more substantial changes in the amide I region and a number of more gradual peak shifts elsewhere (e.g., from 1549 to 1563 cm(-1)), which are not apparent in the MeCbl data. We attribute these differences to interactions between the bulky adenosyl and the corrin ring after photoexcitation and during radical pair recombination, respectively. Although spectrally similar to the initial excited state, the long-lived metal-to-ligand charge transfer state of MeCbl is clearly resolved in the kinetic analysis. The excited states of CNCbl and OHCbl relax to the ground state within 40 ps with few significant peak shifts, suggesting little or no homolysis of the bond between the Co and the upper axial ligand. Difference spectra from density functional theory calculations (where spectra from simplified cobalamins with an upper axial methyl were subtracted from those without) show qualitative agreement with the experimental data. They imply the excited state intermediates in the TRIR difference spectra resemble the dissociated states vibrationally (the cobalamin with the upper axial ligand missing) relative to the ground state with a methyl in this position. They also indicate that most of the TRIR signals arise from vibrations involving some degree of motion in the corrin ring. Such coupling of motions throughout the ring makes specific peak assignments neither trivial nor always meaningful, suggesting our data should be regarded as IR spectral fingerprints.
Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3′–5′ proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3′ base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3′–5′ proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases.
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 O2 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.
Radical control: A single glutamate residue (E287) in the coenzyme B12‐dependent ethanolamine ammonia lyase is pivotal to generating high‐energy radical intermediates during catalysis and then controlling the radical chemistry. Using a dynamic, electrostatic mechanism, it helps to guide the radical intermediates towards the desired end by preventing side reactions.
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