Conformational change accompanies redox reactions of the tetraheme cytochrome c-554 of Nitrosomonas europaea

Abstract

Cytochrome c-554 of the ammonia-oxidizing chemolithoautotropic bacteria is thought to mediate electron transfer from hydroxylamine oxidoreductase to a terminal oxidase and/or to ammonia monooxygenase. The cytochrome has four c hemes which interact magnetically and have the same redox potential.We report that the kinetics of reduction of ferric cytochrome c-554 by dithionite or the oxidation of ferrous cytochrome c-554 by O2 or H 2 0 2 are complex and multiphasic. Transient rapid-scan difference spectra indicate discrete maxima at approximately 418 nm, 425 nm and 432 nm. Absorbance changes at all three difference maxima appear to occur in all kinetic phases, although not in equal amounts for each wavelength.Reduction by 20 mM dithionite was biphasic. At pH 7.5 the first phase, which involved approximately 50% of the total absorbance change, had a rate constant (20°C) of 140 s -l and energy of activation of 20 kJ . mol-'.The slow phase had a rate constant 0.43 sC1 and a relatively high energy of activation, 87 kJ . mol-l, suggesting that a change in protein configuration accompanied the reaction. As the pH of the solution increased, the rate constant for both phases decreased and the fraction of absorbance change in the rapid phase increased. Oxidation of ferrous cytochrome c-554 by O2 involved a discrete rapid phase with a rate constant of 14 s -l , accounting for 6% of the absorbance. The remainder of the reaction was multiphasic with rate constants in the range 0.1 -0.01 s-'. With H 2 0 2 as the oxidant, the rapid phase involved 39% of the change in absorbance with a rate constant of 19 s-'. The remainder of the reoxidation was multiphasic with rate constants ranging over 0.4-0.01 s-'.Oxidation of NH3 to HN02 by Nitrosomonas europaea appears to involve the sequential action of a membrane ammonia monooxygenase (AMO) (NH3 + 2e-+ 2H' + O2 + N H 2 0 H + H20, [I]) and hydroxylamine oxidoreductase (HAO) (NH20H + H 2 0 + HNOz + 4e-+ 4H+, [2]). The latter enzyme is a complex multiheme protein [3] which is reduced by NH20H and is presumed to donate two electrons to the ammonia monoxygenase and two electrons to a membrane terminal oxidase (2H' + 2e-+ 1/2 O2 -, H 2 0 [4]).

Resolution of the four hemes of cytochrome c554 from Nitrosomonas europaea by redox potentiometry and optical spectroscopy

“…Finally, using the < values at 419 nm from Table III, a 2:1 ratio of amplitudes would have been expected on the basis of this revised interpretation. This is reasonably close to the ratio observed in several of the figures presented previously (DiSpirito et al, 1987).…”

“…A previous report investigating the kinetics of reduction of ferric cytochrome c554 by dithionite used rapid-scan absorption spectra in an attempt to differentiate hemes in cytochrome c554 (DiSpirito et al, 1987). The data suggested that there were distinct spectral bands hidden within the unusually broad Soret and a bands reported for this protein.…”

High-resolution structures of the oxidized and reduced states of cytochrome c554 from Nitrosomonas europaea

“…These observations suggest that the observed structural changes are significant and not associated with inherent flexibility of cyt c554 in that region. This conformational change may contribute to the biphasic kinetics observed upon dithionite reduction [25].…”

High-Molecular-Mass Multi- c -Heme Cytochromes from Methylococcus capsulatus Bath

“…Whether the oxidation of hydroxylamine also provides reductant for ammonia (or methane) oxidation or whether all four electrons are transferred to the terminal oxidases (21,62) via cytochrome cЈ (64) and cytochrome c 555 (5) has not been determined for methanotrophs. In the nitrifying bacterium Nitrosomonas europaea, the four electrons from the oxidation of hydroxylamine are transferred to the tetraheme cytochrome, cytochrome c 554 , which acts as a redox mediator from hydrox-ylamine oxidoreductase to both the ammonia monooxygenase and the terminal oxidase (17,33). In the current study, we present the isolation of an octyl-heme cytochrome, cytochrome c 553O .…”