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The reactions of nitric oxide (NO) with the oxidized and reduced forms of fungal and tree laccase, as well as with tree laccase depleted in type 2 copper, are reported. The products of the reactions were determined by NMR and mass spectroscopy, whereas the oxidation states of the enzymes were monitored by EPR and optical spectroscopy. All three copper sites in fungal laccase are reduced by NO. In addition, NO forms a specific complex with the reduced type 2 copper. NO similarly reduces all of the copper sites in tree laccase, but it also oxidizes the reduced sites produced by ascorbate or NO reduction. A catalytic cycle is set up in which N2O, NO2-, and various forms of the enzyme are produced. On freezing of fully reduced tree laccase in the presence of NO, the type 1 copper becomes reoxidized. This reaction does not occur with the enzyme depleted in type 2 copper, suggesting that it involves intramolecular electron transfer from the type 1 copper to NO bound to the type 2 copper. When the half-oxidized tree laccase is formed in the presence of NO, a population of molecules exists which exhibits a type 3 EPR signal. A triplet EPR signal is also seen in the same preparation and is attributed to a population of the enzyme molecules in which NO is bound to the reduced copper of a half-oxidized type 3 copper site.

Oxidized cytochrome c oxidase is shown to exist in three conformations in addition to the transient "g5" conformation previously reported [Shaw, R. W., Hansen, R. E., & Beinert, H. (1978) J. Biol. Chem. 253, 6637-6640]. The "resting" and "g12" conformations are distinguished by an NO-induced cytochrome a3 electron paramagnetic resonance (EPR) signal and an EPR signal at g' = 12, respectively. The "oxygenated" conformatin exhibits an unusual EPR signal in the presence of fluoride and is identical with the "oxygenated" state first discovered by Okunuki et al. [Okunuki, K., Hagihora, B., Sekuzu, I., & Horio, T. (1958) Proc. Int. Symp. Enzyme Chem., Tokyo, Kyoto, 264]. It is proposed that when the reduced enzyme is reoxidized by dioxygen, the oxidized enzyme first relaxes from the "g5" into the "oxygenated" conformation after which a percentage of the molecules slowly relax into the "g12" conformation. The "resting" conformation is not formed when the enzyme is reoxidized. On the basis of the EPR observations, it is proposed that these various conformations of the oxidized enzyme differ in the structure of the cytochrome a3--Cua3 site. Structures for the cytochrome a3--Cua3 site are proposed for each conformation, and a mechanism by which these conformations undergo interconversion among themselves is described.