Small-Molecule Tunnels in Metalloenzymes Viewed as Extensions of the Active Site

Abstract

Conspectus Rigorous substrate selectivity is a hallmark of enzyme catalysis. This selectivity is generally ascribed to a thermodynamically favorable process of substrate binding to the enzyme active site based upon complementary physiochemical characteristics, which allows both acquisition and orientation. However, this chemical selectivity is more difficult to rationalize for diminutive molecules that possess too narrow a range of physical characteristics to allow either precise positioning or discrimination between a substrate and an inhibitor. Foremost among these small molecules are dissolved gases such as H2, N2, O2, CO, CO2, NO, N2O, NH3, and CH4 so often encountered in metalloenzyme catalysis. Nevertheless, metalloenzymes have evolved to metabolize these small-molecule substrates with high selectivity and efficiency. The soluble methane monooxygenase enzyme (sMMO) acts upon two of these small molecules, O2 and CH4, to generate methanol as part of the C1 metabolic pathway of methanotrophic organisms. sMMO is capable of oxidizing many alternative hydrocarbon substrates. Remarkably, however, it will preferentially oxidize methane, the substrate with the fewest discriminating physical characteristics and the strongest C–H bond. Early studies led us to broadly attribute this specificity to the formation of a “molecular sieve” in which a methane- and oxygen-sized tunnel provides a size-selective route from bulk solvent to the completely buried sMMO active site. Indeed, recent cryogenic and serial femtosecond ambient temperature crystallographic studies have revealed such a route in sMMO. A detailed study of the sMMO tunnel considered here in the context of small-molecule tunnels identified in other metalloenzymes reveals three discrete characteristics that contribute to substrate selectivity and positioning beyond that which can be provided by the active site itself. Moreover, the dynamic nature of many tunnels allows an exquisite coordination of substrate binding and reaction phases of the catalytic cycle. Here we differentiate between the highly selective molecular tunnel, which allows only the one-dimensional transit of small molecules, and the larger, less-selective channels found in typical enzymes. Methods are described to identify and characterize tunnels as well as to differentiate them from channels. In metalloenzymes which metabolize dissolved gases, we posit that the contribution of tunnels is so great that they should be considered to be extensions of the active site itself. A full understanding of catalysis by these enzymes requires an appreciation of the roles played by tunnels. Such an understanding will also facilitate the use of the enzymes or their synthetic mimics in industrial or pharmaceutical applications.

The Mechanism of Biochemical NO‐Sensing: Insights from Computational Chemistry

“…and Caldanaerobacter subterraneus, making it highly selective and typical molecular tunnel in H-NOX proteins. [53,55] In addition, we found another longer access tunnel (between helices αA and αD) that gas molecules only rarely used to migrate to the distal pocket. Previously, we identified a third tunnel in H-NOX proteins, [53] however, it is blocked by two aromatic amino acids (His 81 and Tyr 110 ), which form a steric barrier mostly preventing NO molecules from diffusing into the H-NOX protein matrix.…”

The Crystal Structure of Cysteamine Dioxygenase Reveals the Origin of the Large Substrate Scope of This Vital Mammalian Enzyme

“…Computational modeling of peptide-bound ADO shows that substrate RGS5 would likely fill the larger active site tunnel and force the delivery of co-substrate O 2 to proceed through a separate channel. To explore this possibility, MOLE2.5 was used to identify potential tunnels, cavities, and pores. − When the tunnel radius was decreased from the default value of 1.25 Å to a value of 0.49 Å (and using an internal threshold of 0.9 Å and a cutoff ratio of 0.5 while keeping all other parameters at their default values), a tunnel was identified that starts at residues Cys120 and Cys169 and ends at the Fe atom (Figure S9). Thus, our MOLE2.5 analysis corroborates the proposal that this tunnel could selectively allow O 2 to access the substrate-bound active site.…”

Metallocavitins as Promising Industrial Catalysts: Recent Advances

“…Nevertheless, metalloenzymes have evolved to metabolize these small-molecule substrates with high selectivity and efficiency due to small-molecule tunnels and gate-effects. It delivers the right substrate to the right location at the right time, for example, for selective oxidation of the strongest aliphatic hydrocarbon bond in methane" (Banerjee and Lipscomb, 2021). "Also for the small molecule discussed here, the spatial and temporal control of delivery of protons and electrons to the active site is crucial to maintain product selectivity in these transformations" (Amanullah et al, 2021).…”