Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis

Yue Dai,Mohammad Mahfuzul Haque,Dennis J Stuehr

doi:10.1074/jbc.m117.777219

Yue Dai, Mohammad Mahfuzul Haque + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m117.777219

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Abstract

The signaling molecule nitric oxide (NO) is synthesized in animals by structurally related NO synthases (NOSs), which contain NADPH/FAD- and FMN-binding domains. During catalysis, NADPH-derived electrons transfer into FAD and then distribute into the FMN domain for further transfer to internal or external heme groups. Conformational freedom of the FMN domain is thought to be essential for the electron transfer (ET) reactions in NOSs. To directly examine this concept, we utilized a "Cys-lite" neuronal NOS flavoprotein domain and substituted Cys for two residues (Glu-816 and Arg-1229) forming a salt bridge between the NADPH/FAD and FMN domains in the conformationally closed structure to allow cross-domain disulfide bond formation or cross-linking by bismaleimides of various lengths. The disulfide bond cross-link caused a ≥95% loss of cytochrome c reductase activity that was reversible with DTT treatment, whereas graded cross-link lengthening gradually increased activity, thus defining the conformational constraints in the catalytic process. We used spectroscopic and stopped-flow techniques to further investigate how the changes in FMN domain conformational freedom impact the following: (i) the NADPH interaction; (ii) kinetics of electron loading (flavin reduction); (iii) stabilization of open versus closed conformational forms in two different flavin redox states; (iv) reactivity of the reduced FMN domain toward cytochrome c; (v) response to calmodulin binding; and (vi) the rates of interflavin ET and the FMN domain conformational dynamics. Together, our findings help explain how the spatial and temporal behaviors of the FMN domain impact catalysis by the NOS flavoprotein domain and how these behaviors are governed to enable electron flow through the enzyme.

Highlights

The signaling molecule nitric oxide (NO) is synthesized in animals by structurally related NO synthases (NOSs), which contain NADPH/FAD- and FMN-binding domains
Because cross-linking the NADPH/FAD and FMN domains together through either a disulfide bond or a short linker (BMOE) greatly diminished CLSS electron flux to cytochrome c, our results directly demonstrate that freedom of movement of the FMN domain is critical for catalysis of electron flux by nNOSr
Having a stable closed form of CLSS allowed us to measure its electron transfer (ET) and catalytic properties, which otherwise cannot be derived from the dynamic conformational mixtures that normally exist

Summary

To whom correspondence should be addressed

Oxygenase domain (NOSoxy) that catalyzes NO synthesis and a C-terminal flavoprotein or reductase domain (NOSr) that provides electrons and is linked to NOSoxy by an intervening calmodulin (CaM)-binding sequence [3]. We used previously generated Cys-lite nNOSr (CL nNOSr), in which reactive cysteines have been mutated to serines [17], and for site-specific cross-linking studies, we generated a CLSSnNOSr variant by mutating Arg-1229 in the NADPH/FAD domain and Glu816 in the FMN to cysteines in our CL nNOSr construct This allowed us to investigate how restricting FMN domain movement, either tightly or by allowing graded degrees of conformational freedom, would impact the steady-state catalytic activity (cytochrome c reduction) and CaM response of nNOSr, as well as influence the key steps and parameters that underlie its catalysis. Our findings refine our understanding of how FMN domain motion relates to catalysis and regulation in NOSr and, by extension, to other members of the dual-flavin reductase family

Results

Discussion

Experimental procedures