Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea.
During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS)
Arciero
D M
DM
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Balny
C
C
Hooper
A B
AB
D064753:Ammonium Hydroxide; D003574:Cytochrome c Group / Q000378:metabolism*; D004563:Electrochemistry; D006418:Heme / Q000378:metabolism; D006878:Hydroxides; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism; D007700:Kinetics; D009606:Nitrosomonas / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011487:Protein Conformation; D013053:Spectrophotometry; D013816:Thermodynamics
D003574:Cytochrome c Group; D006878:Hydroxides; D006898:Hydroxylamines; C083951:cytochrome c553; D019811:Hydroxylamine; D006418:Heme; D064753:Ammonium Hydroxide; C022302:cytochrome C-552; D010088:Oxidoreductases; C021401:hydroxylamine dehydrogenase