Abstract
It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.
Highlights
While protein−protein complexes are ubiquitous in biology, there are a number of cases where redox partner proteins that are known to interact in a transient manner are found tethered in a single polypeptide chain.[3,4]
Understanding how and why redox partner proteins are tethered in nature and how tethering impacts on enzyme catalysis is of fundamental importance, as well as being key for the development of novel tethered enzymes for use in biocatalytic and synthetic biology applications.[5,6]
With these constituent proteins and their biophysical characterization (Figure S2), we set out to investigate the functional consequences of tethering in RpNiR catalysis
Summary
Understanding how and why redox partner proteins are tethered in nature and how tethering impacts on enzyme catalysis is of fundamental importance, as well as being key for the development of novel tethered enzymes for use in biocatalytic and synthetic biology applications.[5,6] With an increased number of studies on the few known naturally occurring tethered systems (e.g., P450 BM37 and nitric oxide synthase8,9) in recent years, flexible domain tethering has been shown to play a role in reducing the conformational search space and increasing the population of productive electron transfer configurations. Copper-containing nitrite reductases (CuNiRs) are a highly conserved group of enzymes that are naturally found both tethered and separated from their redox partner proteins.[4,10,11] They are highly suited for systematic study to establish the role of redox partner tethering (Figure 1A,B). 2domain CuNiRs catalyze the reduction of NO2− to NO by transferring electrons that originate from the partner proteins (pseudo)azurin (PAz/Az) or cytochrome c (cyt c), through a Received: March 27, 2019 Revised: May 19, 2019 Published: May 29, 2019
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