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)     

A partial resolution and reconstitution of the ammonia-oxidizing system of Nitrosomonas europaea: role of cytochrome c554

The cell-free ammonia-oxidizing system of Nitrosomonas europaea was resolved into three major fractions: a membrane fraction containing cytochrome a1 and c-type cytochromes, a fraction with hydroxylamine-cytochrome c reductase and a cytochrome c fraction. The ammonia-oxidizing activity was reconstituted by the combination of these three fractions. The activity was more consistently reconstituted by adding Nitrosomonas cytochrome c554 to the membrane fraction. The hydroxylamine-cytochrome c reductase activity of the membrane fraction increased with the addition of cytochrome c554, but the oxidation of hydroxylamine to nitrite required a further addition of cytochrome c552. The ammonia oxidation by the membrane plus cytochrome c554 was affected by the concentration of phosphate and the addition of bovine serum albumin, spermine, or MgCl2.     

Catalytic properties of cytochrome c oxidase purified from Nitrosomonas europaea

Cytochrome c oxidase of Nitrosomonas europaea reacts with not only the native cytochrome c (N. europaea cytochrome c-552) but also horse and yeast cytochromes c. The effects on its reactivity of various reagents were very different between the reactions with the native and eukaryotic cytochromes c as the electron donors. The oxidation of eukaryotic ferrocytochrome c by the oxidase was activated by addition of anionic detergents such as sodium dodecyl sulfate and sodium cholate, and anionic phospholipids such as cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine, while the reaction was not activated by Triton X-100, Tween 20, or phosphatidylcholine. However, the reaction with the native cytochrome c of the enzyme was hardly affected by any of the detergents and phospholipids mentioned above, while it was activated by the presence of poly-L-lysine.     

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

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 H2O2 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 degrees C) of 140 s-1 and energy of activation of 20 kJ X mol-1. The slow phase had a rate constant 0.43 s-1 and a relatively high energy of activation, 87 kJ X mol-1, 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-1, accounting for 6% of the absorbance. The remainder of the reaction was multiphasic with rate constants in the range 0.1-0.01 s-1. With H2O2 as the oxidant, the rapid phase involved 39% of the change in absorbance with a rate constant of 19 s-1. The remainder of the reoxidation was multiphasic with rate constants ranging over 0.4-0.01 s-1.     

CO-binding c-type cytochromes and a high-potential cytochrome c in Nitrosomonas europaea.

Resonance-Raman spectra of Japanese-lacquer-tree (Rhus vernicifera) laccase, type-2-copper-depleted laccase and the latter form treated with H2O2 were measured in liquid and frozen solution, on excitation into the 600 nm absorption band. Significant changes in intensity and/or frequency of the bands lying in the 370-430 cm-1 region were observed on freezing, indicating local structural rearrangements taking place at the blue copper site. These findings corroborate previous suggestions based on e.p.r. measurements and redox data [Morpurgo, Calabrese, Desideri & Rotilio (1981) Biochem. J. 193, 639-642]. They show the strong dependence of the physical properties of blue copper centres on local symmetry. Some conclusions on the origin of the Raman bands are also drawn.     

Protonation and inhibition of Nitrosomonas europaea cytochrome c peroxidase observed with protein film voltammetry

The impact of protonation and inhibitor binding of the diheme cytochrome c peroxidase (CCP) from Nitrosomonas europaea has been examined by the technique of catalytic protein film voltammetry (PFV). Previous efforts have shown that the low-potential heme active site (L) binds substrate and yields electrocatalysis at an pyrolytic graphite edge electrode, with properties evocative of a high-potential intermediate, with E(m)>540mV (vs. normal hydrogen electrode) [A.L. Bradley, S.E. Chobot, D.M. Arciero, A.B. Hooper, S. J. Elliott, J. Biol. Chem. 279 (2004) 13297-13300]. Here we demonstrate through similar experiments that catalytic PFV generates limiting currents which allow for electrochemically-detected enzymology of the Ne CCP: such as the demonstration that pH-dependent Michaelis-Menten constants (K(m) values) reveal a pK(a) value of 6.5 associated with the "ES" complex. Further, the direct electrocatalysis is shown in the presence of known inhibitors (cyanide and azide), indicating that inhibitor binding occurs at L, and shifts the resulting catalytic midpoint potential in a negative direction. Michaelis-Menten treatment of the limiting currents generated in the presence of variable concentrations of inhibitors showed that cyanide behaved as a competitive inhibitor with a K(i) value of 0.15muM; azide revealed a mixed-mode of inhibition. The observed data were found to support a previous model of electrocatalysis, and the role of proton transfer chemistry in the active site is discussed in terms of a structural model.     

Chromatography of oxidized and reduced cytochrome c on carboxymethylcellulose

1. Cytochrome c was isolated from horse heart by a chromatographic method. 2. Oxidized and reduced cytochrome c were chromatographed on CM-cellulose that was in equilibrium with several buffer systems of constant composition at pH values of 8.4, 6.75 and 4.9. 3. Separation was better at the higher pH values; the oxidized form was retarded more than twice as much as the reduced form, though they differed by only a single charge. 4. Self-competition between cytochrome molecules is suggested to account for the peak distortion observed at high loads (above 20mum protein concentration).     

Structure of the soluble domain of cytochrome c(552) from Paracoccus denitrificans in the oxidized and reduced states

The crystal structure of the soluble domain of the membrane bound cytochrome c(552) (cytochrome c(552)') from Paracoccus denitrificans was determined using the multiwavelength anomalous diffraction technique and refined at 1.5 A resolution for the oxidized and at 1. 4 A for the reduced state. This is the first high-resolution crystal structure of a cytochrome c at low ionic strength in both redox states. The atomic model allowed for a detailed assessment of the structural properties including the secondary structure, the heme geometry and interactions, and the redox-coupled structural changes. In general, the structure has the same features as that of known eukaryotic cytochromes c. However, the surface properties are very different. Cytochrome c(552)' has a large strongly negatively charged surface part and a smaller positively charged area around the solvent-exposed heme atoms. One of the internal water molecules conserved in all structures of eukaryotic cytochromes c is also present in this bacterial cytochrome c. It contributes to the interactions between the side-chain of Arg36 and the heme propionate connected to pyrrole ring A. Reduction of the oxidized crystals does not influence the conformation of cytochrome c(552)' in contrast to eukaryotic cytochromes c. The oxidized cytochrome c(552)', especially the region of amino acid residues 40 to 56, appears to be more flexible than the reduced one.     

Fast structural dynamics in reduced and oxidized cytochrome c

The sub‐nanosecond structural dynamics of reduced and oxidized cytochrome c were characterized. Dynamic properties of the protein backbone measured by amide ¹⁵N relaxation and side chains measured by the deuterium relaxation of methyl groups change little upon change in the redox state. These results imply that the solvent reorganization energy associated with electron transfer is small, consistent with previous theoretical analyses. The relative rigidity of both redox states also implies that dynamic relief of destructive electron transfer pathway interference is not operational in free cytochrome c.     

Differences in the solution structures of oxidized and reduced cytochrome c measured by small-angle X-ray scattering

While X-ray crystallographic data on cytochrome c show the reduced and oxidized forms to have very similar structures, there is a considerable body of data, mostly from solution studies, that indicates the reduced form is more stable and that the interior of the protein is less accessible to solvent in this state. These observations have led to the hypothesis that while the time-averaged structure is preserved between the two forms, the dynamics of the two forms are different. The oxidized form has been proposed to undergo more large-amplitude, low-frequency motions than the reduced form. The crystal structure data were derived from crystals grown in high salt concentrations, but the solution studies were done at relatively low ionic strength. Small-angle X-ray scattering has been used to examine the effects of the ionic strength and oxidation state on the solution structure of cytochrome c. We find that the radius of gyration and the maximum linear dimension of oxidized cytochrome c are significantly larger than those for reduced cytochrome c, in 5 mM phosphate buffer at pH 7.3, and further that this difference is suppressed by addition of 200 mM sodium chloride. We conclude that there is a real structural difference between the two forms at low ionic strength in solution and that this difference is likely to contribute to the observed differences in accessibility and compressibility.     

N-terminal amino acid sequence of cytochrome c-552 from Nitrosomonas europaea

Nitrosomonas europaea is an ammonia-oxidizing bacterium which contains multiple c-type cytochromes. Few of these components have been assigned physiological roles, but on the basis of molecular weight and redox potential cytochrome c-552 has been considered to be an analogue of the mitochondrial cytochrome-c family of proteins. We present the N-terminal amino acid sequence (47 residues) of cytochrome c-552 and show that this protein is most closely related to the group of small cytochrome-c components from pseudomonads (cytochromes c-551) and is probably evolutionarily distant from the analagous protein (cytochrome c-550) from the nitrite-oxidizing bacterium Nitrobacter agilis.     

The reaction between reduced azurin and oxidized cytochrome c peroxidase from Pseudomonas aeruginosa

The reaction between ferric Pseudomonas cytochrome c peroxidase and reduced azurin was investigated by static titration, rapid scan, and stopped flow techniques as well as circular dichroism measurements. Kinetics of the reduction of the enzyme under pseudo-first order conditions reveals a biphasic logarithmic curve indicating that the reaction between enzyme and azurin is complex and comprises of two reactions, one rapid and a slower one. The relative portion of the fast phase from the overall reaction increases with increasing azurin concentration. Kinetic results can be explained by postulating the presence of two different enzyme forms which are slowly interconvertible. Both enzymatic forms form a complex with reduced azurin. The electron transfer between proteins occurs within the molecular complex of azurin and the peroxidase.     

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c in 0.15M KCl at 20°C have been obtained by timed titration (0.125–0.500 μmol/sec) from the isoionic points (pH 10.2–10.4) to pH 3 and back to the isoionic point. Computer-assisted (PROPHET) data acquisition and blank corrections give curves with good precision with a maximum standard deviation of 0.3 groups for an average error of 1%. The potentiometric titration curve of reduced cytochrome c is reversible within the precision of the method and for the pH range studied. The potentiometric curves for oxidized cytochrome c titrated upscale (pH 3–10) and downscale (pH 10–3) are not reversible. However, they show the same ionization behavior after the initial downscale titration. This is probably the result of a conformational change. Comparison of the data herein reported with the titration curves of oxidized cytochrome c already published by others indicates good agreement on the basis of a normalization of the concentration of protein or on the basis of 25 titrable groups between the acid end point and the isoionic pH. Titration of the 2 μmol imidazole in the upscale or downscale direction gives the correct analytical concentration and pK′ after correction for the solvent titration. Titration of reduced cytochrome c in the presence and absence of an additional equivalent of imidazole gave a difference titration curve, which indicates that a group on the protein shifts from pK′ 5.8 to pK′ 5.3 in the presence of imidazole. The pK′ of imidazole, in the presence of the protein, remains at a nearly normal value of 7.34.     

Cytochrome a1 of Nitrosomonas europaea resembles aa3-type cytochrome c oxidase in many respects

Cytochrome c oxidase of Nitrosomonas europaea has been called cytochrome a₁ by Erickson et al. (Erickson, R.H., Hooper, A.B. and Terry, K.R. (1972) Biochim. Biophys. Acta 283, 155–166) because the reduced form of their preparation had the α peak at 595 nm. In the present studies, the enzyme was purified to an electrophoretically almost homogeneous state and some of its properties were studied. The enzyme much resembled cytochrome aa₃-type oxidase although its reduced form showed the α peak at 597 nm. (1) The absorption spectra of the CO compound of the reduced enzyme and CN⁻ compounds of the oxidized and reduced enzyme were similar to those of the respective compounds of cytochrome aa₃, as well as the absorption spectrum of the intact enzyme resembled that of the cytochrome. (2) The enzyme possessed two molecules of haem a and 1–2 atoms of copper in the molecule. (3) The enzyme molecule was composed of two kinds of subunits of M_r 50000 and 33000, respectively, as are other bacterial cytochromes aa₃. Although the enzyme resembled other bacterial cytochromes aa₃ in many properties, it differed greatly in two properties; its CO compound was easily dissociated into the oxidized enzyme and CO in air, and 50% inhibition of its activity by CN⁻ required approx. 100 μM of the reagent. The enzyme oxidized 0.57, 1.6 and 1.8 mol horse, Candida krusei and N. europaea ferrocytochromes c per s per mol haem a, respectively, in 10 mM phosphate buffer, pH 6.0. The turnover numbers with eukaryotic ferrocytochromes c were increased to 32 and 14, respectively, by addition of cardiolipin (14 μ · ml⁻¹).     

The amino acid sequence of Nitrosomonas europaea cytochrome c-552

The complete amino acid sequence of cytochrome c-552 derived from the chemoautotrophic ammonia-oxidizing bacterium Nitrosomonas europaea was determined. The cytochrome consisted of 81 amino acid residues, and its molecular weight was calculated to be 9098 including heme c. Although the sequence of cytochrome c-552 was highly homologous to those of cytochromes c-551, which were known as the electron-donating components to dissimilatory nitrite reductase in pseudomonads, cytochrome c-552 differed from cytochrome c-551 in two points: (1) the sequence of cytochrome c-552 was shorter by two amino acid residues than that of cytochrome c-551 at the N-terminus and (2) one amino acid insertion was present in cytochrome c-552.