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

Abstract

The electrochemical behavior of tetraheme cytochrome c554 from Nitrosomonas europaea has been studied by thin-layer spectroelectrochemistry, cyclic voltammetry, differential pulse voltammetry, and alternating current voltammetry. Three redox couples were detected. Midpoint potentials for the high-m intermediate-, and low-potential couples are +47, -147, and -276 mV, respectively, from the spectroelectrochemical measurements and +50, -120, and -225 mV, respectively, from the voltammetry measurements. A coulometric titration shows that two electrons are taken up by the high-potential couple and one each is taken up by the intermediate- and low-potential couples. Results from the spectroelectrochemical titration at carefully chosen wavelengths indicate that the intermediate- and low-potential couples obey simple Nernstian behavior. The electrochemical behavior of the high-potential couple is apparently not truly Nernstian but is most consistent with two sites exhibiting slight positive cooperativity. Spectral changes associated with the three couples reveal distinctive features in the reduced-minus-oxidized difference spectra. The difference spectrum of the high-potential pair of hemes suggest a mixture of a high-spin heme and a low-spin heme with maxima at 424 and 432 nm. The difference spectrum of the intermediate-potential heme is low spin with a split Soret with maxima at 414 and 424 nm. The difference spectrum of the low-potential heme also shows a split Soret with maxima at 418 and 432 nm.

Purification and Magneto-optical Spectroscopic Characterization of Cytoplasmic Membrane and Outer Membrane Multiheme c-Type Cytochromes from Shewanella frigidimarina NCIMB400

“…As HAO has been shown by x-ray crystallographic analysis to exist as a trimer (35), and this multimerization is reasoned to be essential for stabilization and catalytic function, the HAO-Cyt c 554 electron transfer complex is thought to be consistent of 36 hemes. Comparable data regarding reduction potential ranges to that presented here for the membranous c-hemes of S. frigidimarina NCIMB400 have also been obtained for the HAO (36) and Cyt c 554 (37) of N. europea, with the lowest midpoint values noted for these proteins being Ϫ390 and Ϫ226 mV, respectively. Furthermore, x-ray crystallography has revealed a number of structural similarities between the tetraheme core of Cyt c 554 (38) and four of the c-hemes of HAO (35).…”

In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper

“…Under optimal conditions, their extracts catalyzed ammonia-dependent 02 uptake at rates close to 10% of the rates observed with whole-cell suspensions (48). The extracts were, however, quite unstable: upon storage at 40C, the extracts lost 50% of their AMO activity in 4 h, and when frozen at 20'C, they were stable for only 1 to 2 days. The activity of the extracts was also variable and dependent upon the age and in vivo AMO activity of the N. europaea cells.…”

“…These studies were expanded to demonstrate that purified, reduced cytochrome c554 could serve as a reductant for the oxidation of ammonia or CO by the membrane fraction, leading to the suggestion that cytochrome c554 may serve as a direct electron donor to AMO (51). Recently, it has been shown that cytochrome c554 will also form a stable complex with and serve as an electron acceptor for purified HAO (3,4 Ba"4CO3 (specific activity = 56 mCi/mmol), obtained from Sigma Chemical Co., St. Louis, Mo., as described previously (19). Unlabelled acetylene was generated in a gasgenerating bottle from calcium carbide (Aldrich Chemical Co., Milwaukee, Wis.) as described previously (18).…”

Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea

“…NH3 + 02+ 2e-+ 2H+ -> NH2OH + H20 Next, hydroxylamine is oxidized to nitrite in a dehydrogenase reaction (2) in the periplasm, which is catalyzed by the multiheme enzyme HAO (15) in concert with the tetraheme electron acceptor cytochrome C554 (3,4):…”