Structural basis of biological NO generation by octaheme oxidoreductases - PubMed (original) (raw)

Hydroxylamine and hydrazine oxidation by kustc1061 and NeHAO. A, cytochrome c reduction (50 μ

m

) catalyzed by kustc1061 (200 ng) in the presence of 1.5 μ

m

hydroxylamine (blue) or 1.5 μ

m

hydrazine (red). The oxidation of hydroxylamine (1.5 μ

m

) coupled to the reduction of cytochrome c (50 μ

m

) by NeHAO (30 ng) is represented by the green curve. Reactions were followed and quantified by measuring the increase in the absorption at 550 nm (Δϵ550 = 19,600

m

−1 cm−1) as the result of cytochrome c reduction. B, hydroxylamine oxidation (9 μ

m

) by 1.4 μg of kustc1061 (blue) and hydrazine oxidation (9 μ

m

) by 20 μg of kustc1061 (red) recorded by measuring the reduction of cytochrome c at 550 nm (Δϵ550 = 19,600

m

−1 cm−1, right axis). C, NO production from hydroxylamine (8 μ

m

) by kustc1061 (150 ng) in the presence of 50 μ

m

cytochrome c. NO formation was followed by recording the increase in fluorescence as the result of the formation of the nitrosylated derivative of Cu(II)FL2E and was quantified from a calibration curve prepared from known NO stock solutions. D, hydroxylamine oxidation (2 μ

m

) recorded by the increase in fluorescence resulting from the formation of a nitrosylated derivative of the NO-probe 4-amino-5-methylamino-2′,7′-difluorofluorescein (black) and cytochrome c reduction under the same conditions (blue), showing a 1:3 stoichiometry of NO production versus cytochrome c reduction. E, accumulation of 30N2 during [15N]hydrazine oxidation. 15N15N (open squares) was produced upon addition of 7.5 μ

m

H15N-15NH to 2 ml of 20 m

m

potassium Pi buffer containing 1.3 μg of kustc1061 and 50 μ

m

oxidized cytochrome c. The concentration of reduced cytochrome c was determined as in A. NO in C and D was quantified from calibration curves prepared from known NO stock solutions.