Enzymes of aerobic respiration on iron.

Bacteria capable of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Comparative spectroscopic analyses revealed that phylogenetically distinct organisms expressed copious quantities of spectrally distinct redox-active biomolecules during autotrophic growth on soluble iron. Thiobacillus ferroxidans, Leptospirillum ferrooxidans, Sulfobacillus thermosulfidooxidans, and Metallosphaera sedula possessed iron respiratory chains dominated by a blue copper protein, a novel red cytochrome, a novel yellow protein, and a novel yellow cytochrome, respectively. Further investigation of each type of respiratory chain will be necessary to deduce the advantages and disadvantages of each.

A potentiometric and kinetic study on the respiratory chain of ferrous-iron-grown Thiobacillus ferrooxidans.

The type and number of respiratory chain components present in membranes of Thiobacillus ferrooxidans have been investigated. These redox components were resolved potentiometrically and kinetically. Using optical techniques two cytochromes a1, multiple cytochromes c and two cytochromes b were detected. By using electron paramagnetic resonance, two copper-containing centres, high and low spin ferric haems and a ferredoxin centre were detected. Based on the combination of a potentiometric resolution and a kinetic study a model for the sequence of the respiratory chain components in the Fe2+ to O2 branch of the T. ferrooxidans respiratory chain is proposed.

Angular dependence of electron paramagnetic resonances of an azide-NO complex of cytochrome c oxidase: orientation of the haem-copper axis in cytochrome aa3 from ox heart.

The orientation dependence of the EPR signals arising from the azide-nitric oxide complex of cytochrome oxidase was investigated using oriented multilayers of mitochondrial membranes from ox heart. Variations in line shape of the DeltaMS=1 signal of the triplet state were apparent, whilst the DeltaMS=2 transitions between g=4.7 and 3.9 varied in intensity as the angle of the applied magnetic field was varied. These half-field signals were maximal with the field parallel to the membrane plane. A model of the bi-liganded azide-nitric oxide complex has been constructed, in which the nitric oxide is bound to the high-spin haem in a bent configuration, with the Fe-N=O plane at 60-90 degrees to the membrane plane and the azide bound to the copper, distal from the haem. In addition, angular variations of the signals at g'=11 and g' around 3.5, derived from an integer-spin complex, were also observed.

EPR studies of higher plant mitochondria. I Ubisemiquinone and its relation to alternative respiratory oxidations.

An EPR investigation of the region of the higher plant respiratory chain involving ubiquinone and Center S-3 of succinate dehydrogenase is reported. At temperatures close to those of liquid helium, first derivative spectra corresponding to Center S-3 (gmax = 2.017) and a signal split around g = 2.00 (major features of peaks and troughs at g values of 2.045, 2.03, 1.985, 1.97 and 1.96) were observed in mung bean (Phaseolus aureus), Arum maculatum spadix, Sauromatum guttatum spadix and tulip bulb (Tulipa gesnerana) mitochondria. The split signal was small or absent in potato tuber and Symplocarpus foetidus spadix mitochondria. The redox behavior of these signals in mung bean mitochondria in a variety of respiratory steady-state conditions suggested that the components giving rise to them were an integral part of the respiratory chain and were located on the substrate side of coupling Site II. The split signal could be removed by addition of hydroxamic acids in all tissues tested, although the Ks of this effect was an order of magnitude higher than the Ki of inhibition of the alternative respiratory pathway in mung bean and Sauromatum guttatum spadix mitochondria. The results are discussed in relation to the current ideas on the ordering of components in the region around the classical Site II of the respiratory chain and in relation to the location of the alternative respiratory oxidase pathway of higher plants.

Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations.

The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa3-type cytochrome c oxidase. The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a3) and copper (CuB). Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo. Cyanide, sulfide, and azide react with the Fe(3+)-Cu+ center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa3. Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand. Cyanide and azide react with the Fe(3+)-Cu2+ center to yield forms observable via UV-visible spectroscopy but not EPR. With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa3. These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet. Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa3. This suggests that the binuclear centers of the two enzymes are quite similar.

Ligand-binding properties and heterogeneity of cytochrome bo from Escherichia coli.

Cyanide and formate induce spectral changes in E. coli cytochrome bo which are similar to those induced in bovine heart cytochrome-c oxidase (cytochrome aa3). Cyanide induces a red shift of 6 nm in the Soret band, whereas formate induces a blue shift of 2 nm. Cytochrome bo as purified shows multiphasic cyanide-binding kinetics. At least three phases can be seen with rate constants of 16, 1 and 0.1 M-1 s-1, respectively, at pH 7 and 20 degrees C. The enzyme after redox cycling ('pulsing') or in situ in E. coli membranes shows essentially monophasic binding with a rate constant of 15 M-1 s-1. Further evidence of heterogeneity in the enzyme as prepared comes from formate binding, which also shows at least three phases (rate constants of 1.4, 0.2 and 0.01 M-1 s-1, respectively, at pH 5 and 20 degrees C). The fast phase of cyanide binding is eliminated in less than 2 min by incubation with 40 mM formate, but the intermediate phase is unaffected by incubation for 3.5 h with 40 mM formate. Thus, the subpopulation that causes the fast phase of cyanide binding also causes the fast phase of formate binding. Formate-ligated cytochrome bo has similar cyanide-binding kinetics to the subpopulation that causes the slow phase of cyanide binding in cytochrome bo as prepared. It appears, from all this, that the subpopulations responsible for the fast and slow phase of cyanide binding are analogous to the 'fast' and 'slow' forms, respectively, of cytochrome aa3.(ABSTRACT TRUNCATED AT 250 WORDS)

Potentiometric titration of cytochrome-bo type quinol oxidase of Escherichia coli: evidence for heme-heme and copper-heme interaction.

The cytochrome-bo quinol oxidase of Escherichia coli contains a high-spin b-type heme (cytochrome o), a low-spin b-type heme (cytochrome b) and copper. The EPR signal from cytochrome o is axial high spin and when titrated potentiometrically gives a bell-shaped curve. The low-potential side of this curve (Em7 approx. 160 mV) corresponds to the reduction/oxidation of the cytochrome. The high-potential side (Em7 approx. 350 mV) is proposed to be due to reduction/oxidation of a copper center; in the CuII form tight cytochrome o-copper spin coupling results in a net even spin system and loss of the EPR spectrum. Optical spectra of the alpha-bands of the reduced cytochromes at 77 K show that cytochrome b has its maxima at 564 nm when cytochrome o is oxidized but that this shifts to 561 nm when cytochrome o (max. 555 nm) is reduced. Both a heme-copper (cytochrome o-CuII) and a heme-heme (cytochrome o-cytochrome b) interaction are indicated in this quinol oxidase. These results indicate that cytochrome-bo quinol oxidase has a binuclear heme-copper catalytic site and suggest striking structural similarity to subunit I of the cytochrome aa3 system.

Characterisation of a near infra-red absorption band of the Escherichia coli quinol oxidase, cytochrome o, which is attributable to the high-spin ferrous haem of the binuclear site.

The bacterial quinol oxidase, cytochrome o, is an enzyme which is highly analogous to the better known cytochrome c oxidase, cytochrome aa3, but with the important difference that it lacks the near infra-red absorbing pigment CuA. In this article we report an absorption band in the near IR spectrum of cytochrome o with a maximal absorption at 758 nm, and which is attributable to the ferrous high-spin haem. The 758 nm band has an extinction coefficient of 0.2-0.3 mM-1.cm-1 at 758-800 nm. This region in cytochrome aa3 is dominated by the CuA absorption. The 758 nm absorption is lost on addition of CO or cyanide to the reduced enzyme. The carbon monoxide compound of cytochrome o also has absorbance bands in the near infra-red, and these may be attributable to a low-spin ferrous haem compound.

Detection of a near infra-red absorption band of ferrohaem a3 in cytochrome c oxidase.

We have detected weak absorption bands in the near infra-red region of reduced mammalian cytochrome c oxidase, analogous to those that we have recently reported to be present in the bacterial cytochrome o (Ingledew, W.J., Bacon, M. and Rich, P.R. (1992) FEBS Lett. 305, 167-170). The major band is centred at 784 nm and has an sigma mM-1.cm-1 of around 0.1. It is shifted to 760 nm in the carbon monoxide compound and is absent in the reduced cyanide complex. We attribute it to a charge transfer band of ferrohaem a3, equivalent to the 'band III' or 'conformational band' of haemoglobin.

EPR spectroscopy of Escherichia coli cytochrome bo which lacks CuB.

The spectroscopic and ligand-binding properties of a copper-deficient cytochrome bo3, a member of the haem-copper superfamily of terminal oxidases, are reported and contrasted with those of the native enzyme. The enzyme lacks the copper atom (CuB) which is normally an integral part of the catalytic site. The consequences of loss of the CuB are the loss of antiferromagnetic coupling to the high-spin haem and an inability to form any of the integer-spin derivatives of the enzyme. Low-spin compounds of the normally high-spin haem are still formed with appropriate ligands, although these are modified.

EXAFS of the type-1 copper site of rusticyanin.

Extended X-ray absorption fine structure (EXAFS) spectra at the Cu K-edge have been recorded of the oxidized and reduced form at pH 3.5 of rusticyanin, the type-1 or 'blue'-copper protein from Thiobacillus ferrooxidans. The EXAFS of oxidized rusticyanin is well simulated with models assuming a ligand set of 2 N(His) and 1 S(Cys) at 1.99 and 2.16 A, respectively. Upon reduction, the average Cu-N ligand distance increases by approx. 0.08A. For both redox states studied, the fit by the simulation is significantly improved by including a contribution of an additional sulfur ligand at approx. 2.8 A. From comparison with structural data of other blue-copper proteins, it is concluded that the copper coordination environment is relatively rigid, which may be a clue to its high redox potential.

The structural gene for rusticyanin from Thiobacillus ferrooxidans: cloning and sequencing of the rusticyanin gene.

The rusticyanin gene from the acidophilic chemolithotroph Thiobacillus ferrooxidans has been cloned and sequenced. A central portion of the gene was identified by PCR reactions utilising primers optimised for codon bias followed by nested PCR with degenerate primers. The 5' and 3' ends of the rusticyanin gene were then cloned using degenerate primers to each end and anchor sequences to the known internal sequence. The entire gene was amplified using Tli DNA polymerase and specific primers to the 5' and 3' ends and the sequence confirmed after cloning into Bluescript and transformation of XL-1 Blue Escherichia coli.