[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) is a signal molecule produced in animals by three different NO synthases. Of these, only NOS I (neuronal
nitric-oxide synthase; nNOS) is expressed as catalytically active N-terminally truncated forms that are missing either an
N-terminal leader sequence required for protein-protein interactions or are missing the leader sequence plus three core structural
motifs that in other NOS are required for dimer assembly and catalysis. To understand how the N-terminal elements impact nNOS
structure-function, we generated, purified, and extensively characterized variants that were missing the N-terminal leader
sequence (Δ296nNOS) or missing the leader sequence plus the three core motifs (Δ349nNOS). Eliminating the leader sequence
had no impact on nNOS structure or catalysis. In contrast, additional removal of the core elements weakened but did not destroy
the dimer interaction, slowed ferric heme reduction and reactivity of a hemedioxy intermediate, and caused a 10-fold poorer
affinity toward substrate l-arginine. This created an nNOS variant with slower and less coupled NO synthesis that is predisposed to generate reactive
oxygen species along with NO. Our findings help justify the existence of nNOS N-terminal splice variants and identify specific
catalytic changes that create functional differences among them.
[Show abstract][Hide abstract] ABSTRACT: We are combining stopped-flow, stop-quench, and rapid-freezing kinetic methods to help clarify the unique redox roles of tetrahydrobiopterin (H(4)B) in NO synthesis, which occurs via the consecutive oxidation of L-arginine (Arg) and N-hydroxy-L-arginine (NOHA). In the Arg reaction, H(4)B radical formation is coupled to reduction of a heme Fe(II)O(2) intermediate. The tempo of this electron transfer is important for coupling Fe(II)O(2) formation to Arg hydroxylation. Because H(4)B provides this electron faster than can the NOS reductase domain, H(4)B appears to be a kinetically preferred source of the second electron for oxygen activation during Arg hydroxylation. A conserved Trp (W457 in mouse inducible NOS) has been shown to influence product formation by controlling the kinetics of H(4)B electron transfer to the Fe(II)O(2) intermediate. This shows that the NOS protein tunes H(4)B redox function. In the NOHA reaction the role of H(4)B is more obscure. However, existing evidence suggests that H(4)B may perform consecutive electron donor and acceptor functions to reduce the Fe(II)O(2) intermediate and then ensure that NO is produced from NOHA.
[Show abstract][Hide abstract] ABSTRACT: We cloned, expressed, and characterized a hemeprotein from Deinococcus radiodurans (D. radiodurans NO synthase, deiNOS) whose sequence is 34% identical to the oxygenase domain of mammalian NO synthases (NOSoxys). deiNOS was dimeric, bound substrate Arg and cofactor tetrahydrobiopterin, and had a normal heme environment, despite its missing N-terminal structures that in NOSoxy bind Zn(2+) and tetrahydrobiopterin and help form an active dimer. The deiNOS heme accepted electrons from a mammalian NOS reductase and generated NO at rates that met or exceeded NOSoxy. Activity required bound tetrahydrobiopterin or tetrahydrofolate and was linked to formation and disappearance of a typical heme-dioxy catalytic intermediate. Thus, bacterial NOS-like proteins are surprisingly similar to mammalian NOSs and broaden our perspective of NO biochemistry and function.
Proceedings of the National Academy of Sciences 02/2002; 99(1):107-12. DOI:10.1073/pnas.012470099 · 9.67 Impact Factor