Tateo Yamanaka

The oxidation mechanisms of thiosulphate and sulphide in Chlorobium thiosulphatophilum: Roles of cytochrome c-551 and cytochrome c-553

Abstract A thiosulphate-cytochrome c reductase was highly purified from Chlorobium thiosulphatophilum and its properties were studied. The enzyme catalyses reduction with Na 2 S 2 O 3 of c cytochromes, including cytochrome c -551 of the bacterium. Cytochrome c (555, C. thiosulphatophilum ) does not react directly with the enzyme at an appreciable rate but stimulates greatly the reduction by the enzyme of cytochrome c -551 with Na 2 S 2 O 3 . The reduction of c cytochromes catalysed by the enzyme is strongly inhibited by cyanide and sulphite. Cytochrome c (553, C. thiosulphatophilum ), a c -type cytochrome with covalently bound flavin, was found to catalyse reduction with sulphide of c cytochromes, including cytochrome c -555. The reaction is strongly inhibited by cyanide. Cyanide seems to combine strongly with cytochrome c -553 probably at the flavin moiety. Thus, the absorption spectrum attributable to flavin of the haemoprotein is changed on addition of cyanide, and neither the original spectrum nor the activity reappears even after the cyanide-treated cytochrome has been subjected to gel filtration with a Sephadex G-25 column or to isoelectric focusing.

Cytochrome c (553, Chlorobium thiosulfatophilum) is a sulphide‐cytochrome c reductase

T h r e e k inds o f c-Wpe w m c h r o m e s , ¢y tochzome~ e-551 , c-553 a nd e.555 have been isolated f r o m the green zulFhur b a c t e r i u m , ,L~lorobh~na t]~oaTglfa~ophitum I1,2]. Recen*ly , we have succeeded in .~the pur i f i ca i ion of a th iosu]phate-cy toch~orne c reduc.Ia~e der ived fro:m C. ~hiosulfatophilura and f o u n d ~aat cyt o c h r o m e c-551 func t i ons as the d i rec t e ] e c ~ o n ae~-ep~tor fo r the e n z y m e [ 3 ] . Mo]ecu,]ar ~eatnres of eft.oc h r o m e c -553 have been wel~ s t o n e d [ 1 , 4 ] ; i t has flavin (p r c ba b ly F]VlN) as we1] as h a e m c. t towever~ its func,l:i,on was ,obscure. In ~ e p l e sen l invest igat ion, we have found t h a t ~v t cch /0me ,c-553 is ~educed m o r e ~apidly vAT~ su]phide ,1hart o t h e r c y t o c h r o m e s c , and i t ca ta lyses r e d u c t i o n ~ff ey~oehromes c ~Ath sulp.r~de. T h e r e f o r e . c y t o c h r o m e c-553 seems to ac t as a sn lpk ide -cy tochro rne c redue tase . F u r t h e r , we have found *daat ~ e cy to c h r o m e c om bines wi th cy~a ide ve ry s t rong ly an d a~ a xe~ult fla.e ab~o~p*ion s p e c t r u m a tu~but~Me ~o the b o u n d iiavin o f the ~ o c ~ _ r o m e is cons ide rab ly changed.

A high-potential nonheme iron protein (HiPIP)-linked, thiosulfate-oxidizing enzyme derived fromChromatium vinosum

A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was inferred to be tetrathionate.

The pH dependence of the resonance raman spectra and structural alterations at heme moieties of various c-type cytochromes.

The pH dependence of resonance Raman spectra were studied for ferrous and ferric cytochromes c, c2, c3, c-551, and c-555. The frequencies of the 1565 cm-1 (ferric) and 1539 cm-1 lines (ferrous) were sensitive to the replacement of the sixth ligand. The titration curve for the 1565 cm-1 line of cytochrome c was parallel with that for the 695 nm band. The pH dependence of the 1539 cm-1 line of ferrous cytochrome c3 suggested the stepwise replacement of the sixth ligand of its four hemes, although such pH dependence was not recognized for the Raman spectra of other ferrous cytochromes investigated. The relative intensities of three Raman lines at 1639, 1587, and 1561 cm-1 of ferric protoporphyrin bis-imidazole complex were changed clearly by the presence of detergents. The relative intensities of the corresponding three Raman lines of cytochromes b5 and c were close to those of the ferric porphyrin complex in the presence and absence of detergents, respectively, suggesting an appreciable difference in their heme environments. Reduced hemin in detergent solution, unexpectedly, gave the Raman spectrum of ferric low spin type.

Cytochrome a-type terminal oxidase derived from Thiobacillus novellus. Molecular and enzymatic properties

Cytochrome a-type terminal oxidase was purified from Thiobacillus novellus to an electrophoretically homogeneous state and some of its properties were studied. The enzyme shows absorption peaks at 428 and 602 nm in the oxidized form, and at 442 and 602 nm in the reduced form. The CO compound of the reduced enzyme shows peaks at 431 and 599 nm. The enzyme has 1 mol of haem a and 1 g-atom of copper per 55600 g and is composed of two kinds of subunit, of 32000 and 23000 daltons, respectively. The enzyme reacts rapidly with tuna, bonito and yeast cytochromes c as well as with T. novellus cytochrome c, while it reacts slowly with horse and cow cytochromes c. The reduction product of oxygen catalysed by the enzyme is water.

A cytochrome cc′-like haemoprotein isolated from Azotobacter vinelandii

Abstract From Azotobacter vinelandii , a cytochrome cc′ -like haemoprotein has been obtained. It shows an absorption peak at 397 nm in the oxidized form. On reduction with Na2S2O4, a peak appears at 423 nm and a broad band around 550 nm. The reduced form combines with CO, whereas the oxidized form combines with NO. In an alkaline solution, the absorption spectrum of the haemoprotein changes into the haemochrome-type. Although the haemoprotein is autoxidizable, it does not show cytochrome oxidase activity with cytochrome c 4 or c 5 as the electron donor. No biological function of the haemoprotein has been found so far.

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

Abstract A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP+ or NAD+ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP+) is better electron donor (or acceptor), while NADH (or NAD+) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP+, 2′-AMP and and atebrin.

Effect of cardiolipin on the enzymatic activity of Nitrobacter agilis cytochrome c oxidase

Abstract Effects of cardiolipin on the reaction rates of Nitrobacter agilis cytochrome c oxidase with cytochrome c were studied at various concentrations of phosphate buffer. Cardiolipin stimulated greatly the oxidation by the enzyme of horse and yeast ferrocytochromes c, especially at higher ionic strengths. However, the oxidation by the enzyme of N. agilis ferrocytochrome c-550, the physiological electron donor for the oxidase, was not accelerated by addition of cardiolipin. Analysis of the lipid compositions showed that neither the cell membranes of N. agilis nor the enzyme preparation contained cardiolipin. These results suggest that cardiolipin is not necessary for the reaction of N. agilis cytochrome c oxidase with N. agilis cytochrome c-550. On the basis of these results, the difference in the reactivity with cytochrome c of cytochrome c oxidase between the bacterial and mitochondrial enzymes is discussed.

Thiobacillus novellus cytochrome oxidase can separate some eucaryotic cytochromes c

There is a distinct biological specificity in the reaction of certain redox enzymes with cytochrome c and it reflects very well the evolutionary relationship of the organism from which cytochrome c is isolated [l-3]. Most eucaryotic cytochromes c react very rapidly with cow cytochrome oxidase (EC 1.9.3.1) and yeast cytochrome c pcroxidase (EC 1.11.1.5) but react very poorly with Pseudomonas aeruginosa nitrite reductzise (previously called Pseudomonas cytocbrome oxidase, EC 1.9.3.2), while many procaryotic cytochromes c react very poorly with the oxidase and the peroxidase but react rapidly with the reductase. In the previous studies [ 11, we have predicted that there may occur cytochrome oxidases which are situated between the reductase and the oxidase in terms of the reactivity with cytochromes c. In the present investigation, we purified cytochrome oxidase from Thiobacillus novellus and determined its specificity for cytochrome c as the electron donor. The bacterial oxidase has haem a as the prosthetic group and its absorption spectrum is very similar to that of mammalian cytochrome oxidase. However, its specificity for cytochrome c is considerably different from that of cow cytochrome oxidase.

A cytochrome c peroxidase isolated from Thiobacillus novellus

Abstract A cytochrome c peroxidase (ferrocytochrome c :H 2 O 2 oxidoreductase, EC 1.11.1.5) was isolated and highly purified from Thiobacillus novellus , and its properties were studied. The enzyme has haem c as the prosthetic group, and shows a peak at 398 nm in the oxidized form and peaks at 415, 520 and 550 nm in the reduced form. It peroxidatically oxidizes the reduced form of cytochrome c (550, Thiobacillus novellus ) and mammalian-type cytochromes c but does not react or reacts very poorly with bacterial c -type cytochromes such as cytochrome c (551, Pseudomonas aeruginosa ), cytochrome c (552, Nitrosomonas europaea ) and cytochrome c (555, Chlorobium thiosulfatophilum ). One mole of the enzyme oxidizes peroxidatically 2450 moles of tuna ferrocytochrome c per min at pH 8.5 and at 18 °C. The peroxidase reaction catalysed by the enzyme is strongly inhibited by cyanide and azide.