Distinct conformational behaviors of four mammalian dual‐flavin reductases (cytochrome P450 reductase, methionine synthase reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles

Abstract

Multi-domain enzymes often rely on large conformational motions to function. However, the conformational setpoints, rates of domain motions, and relationships between these parameters and catalytic activity is not well understood. To address this, we determined and compared the conformational setpoints and the rates of conformational switching between closed unreactive and open reactive states in four mammalian di-flavin NADPH oxidoreductases that catalyze important biological electron transfer reactions: cytochrome P450 reductase (CPR), methionine synthase reductase (MSR), and endothelial and neuronal NO synthase (eNOS & nNOS). We used stopped-flow spectroscopy, single turnover methods, and a kinetic model that relates electron flux through each enzyme to its conformational setpoint and its rates of conformational switching. Results show that the four flavoproteins, when fully-reduced, have a broad range of conformational setpoints (from 12 to 72% open state) and also vary 100-fold regarding their rates of conformational switching between unreactive closed and reactive open states (CPR > nNOS > MSR > eNOS). Furthermore, simulations of the kinetic model could explain how each flavoprotein can support its given rate of electron flux (cytochrome c reductase activity) based on its unique conformational setpoint and switching rates. Our study is the first to quantify these conformational parameters among the di-flavin enzymes, and suggests how the parameters might be manipulated to speed or slow biological electron flux.

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

“…Because previous ensemble studies could only report on the averaged protein conformational distributions, and could not provide insight into the conformational dynamics (34,37,38,40), our study represents a significant advance and provides an interesting perspective on how CaM may regulate nNOSr electron transfer reactions through its control of protein conformational behavior.…”

“…In the case of endothelial NOS, previous studies and modeling (34,38) would predict that it may exhibit significant differences in its conformational states distribution (favoring more closed states relative to nNOSr) and in its conformational dwell times (favoring longerlived states relative to nNOSr). These differences and related aspects can now be addressed using our single molecule approach.…”

“…The cytochrome c reductase activity was determined at room temperature by monitoring the absorbance increase at 550 nm and using an extinction coefficient e 550 = 21 mM −1 cm −1 as described (34,38). To overcome a dilution-based dissociation of FMN from the nNOSr, we added 6-OH-FMN, which has much less fluorescence than FMN but still binds within nNOSr and supports its catalysis.…”

“…Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.…”

“…In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25,28,32,34,35,37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38)(39)(40)(41)(42).NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25,27).…”