Abstract：
:The inducible nitric oxide synthase core oxygen domain (iNOS(COD)) is a homodimeric protein complex of ca. 100 kDa. In this work, the subunit disassembly and unfolding of the protein following a pH jump from 7.5 to 2.8 were monitored by on-line rapid mixing in conjunction with electrospray (ESI) time-of-flight mass spectrometry. Various protein species become populated during the denaturation process. These can be distinguished by their ligand binding behavior, and by the different charge states that they produce during ESI. Detailed intensity-time profiles were obtained for all of these species, and the kinetics were subjected to a global analysis which allows a model of the denaturation process to be developed. The data are described well by three relaxation times (tau(1) = 0.36 s, tau(2) = 0.62 s, and tau(3) = 3.3 s), each of which has a characteristic amplitude spectrum. The initial step of the reaction is the disruption of the iNOS(COD) dimer, to generate heme-bound monomeric species in various degrees of unfolding. This first step is accompanied by the loss of two tetrahydrobiopterin cofactors. Subsequent heme loss generates monomeric apoproteins exhibiting various degrees of unfolding. In addition, the formation of proteins that are bound to two heme groups is observed. A subpopulation of holo monomers undergoes substantial unfolding while retaining contact with the heme cofactor. Together with previous studies, the results of this work suggest that the occurrence of complex reaction mechanisms involving several short-lived intermediates is a common feature for the denaturation of large multiprotein complexes.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Wilson DJ,Rafferty SP,Konermann Ldoi
10.1021/bi047684ysubject
Has Abstractpub_date
2005-02-22 00:00:00pages
2276-83issue
7eissn
0006-2960issn
1520-4995journal_volume
44pub_type
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