Studies on the metabolism of a sulfur-oxidizing bacterium VII. Oxidation of sulfite by a cell-free extract of Thiobacillus thiooxidans

Properties of the cell-free extract, prepared from a strainof Thiobacillus thiooxidans by sonic disruption followed byfractionation with centrifugatiori, were investigated with referenceto its sulfite-oxidizing activity. Without the addition of cofactors the particulate fraction(F-P)catalyzed oxidation of sulfite with oxygen or bacterial cytochromec-552 obtained from Pseudomonas stutzeri as electron acceptor.TMPD reduced by ascorbic acid was also oxidized by F-P. Thesoluble fraction(F-S) showed no activity in oxidizing sulfiteand TMPD, but stimulated TMPD oxidation by F-P. Oxygen uptake with either sulfite or TMPD as substrate was inhibitedby KCN, NaN₃, CO and c-phenanthroline. CO-Inhibition was reversedby light. Reduction of cytochrome c-552 by sulfite was insensitiveto these agents. Antimycin A markedly inhibited sulfite oxidation with eitheroxygen or cytochrome c-552 as electron acceptor, but was withouteffect on TMPD oxidation. DDC and SAO, both strong inhibitors of sulfur oxidation, didnot affect sulfite and TMPD oxidations. Cytochromes of the a, b and c types were contained in F-P. Thesecytochromes were rapidly reduced when F-P was incubated withsulfite. Cytochrome(s) of the c type was present in F-S, too. ¹VI.=References (3) ²Partly supported by a grant from the Ministry of Education ³Present address: Sanyo Women's College, Hatsukaichi, Hiroshima738, Japan ⁴Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima 734, Japan (Received May 15, 1970; )     

Studies on the metabolism of a sulfur-oxidizing bacterium VI1. Fractionation and reconstitution of the elementary sulfur-oxidizing system of Thiobacillus thiooxidans

The sulfur-oxidizing system of a strain of Thiobacillus thiooxidanswas obtained in cell-free state. The system is resolved intothree fractions and can be reconstituted from these fractions.Both the soluble and particulate fractions are required forthe oxidation of elementary sulfur. The soluble fraction wasfurther separated into two fractions, the collodion membrane-permeable(S-P)and the impermeable(S-IP). S-P contains a low molecular weight,relatively heat stable substance(s) which is indispensable forthe reconstitution of the sulfur-oxidizing system and was identifiedas a pyridine nucleotide. The function of S-P can be replacedby NAD or NADP, but not by cysteine nor GSH. Oxidation of NADH₂ and NADPH₂ is catalyzed by the particulatefraction. Oxidation of the latter is much more rapid than thatof the former. Oxidation of NADPH₂ as well as sulfur oxidationis inhibited by cyanide, pCMB and CO, the CO-inhibition beingphoto-irreversible. However, strong inhibitors of sulfur oxidationsuch as DDC, 8-hydroxyquinoline and salicylaldoxime have noeffect on the oxidation of NADPH₂. The optimum pH values for sulfur and sulfite oxidations by thecell-free extract are shifted to the neutral side in comparisonwith pH values by intact cells. ¹V = References(I). ²Partly supported by a grant from the Ministry of Education. (Received April 3, 1969; )     

Studies on the metabolism of a sulfur-oxidizing bacterium IV.1 Growth and oxidation of sulfur compounds in Thiobacillus thiooxidans

By immersing a few small cellophane bags containing BaCO³ powderin STARKEY's medium, the duration of lag phase in the growthof Thiobacillus thiooxidans is minimized and the yield of cellsis increased ten times that of the previous method. The activitiesof oxidation for sulfur and sulfite change with growth. Sulfiteis oxidized at a comparable rate to that of sulfur oxidationat pH values between 6.0 and 6.5. In the presence of cysteineor glutathione, thiosulfate can be oxidized at a pH above 5.0.At pH values below 4.5, apparent oxidation of thiosulfate andtetrathionate to sulfate is observed. This result is accountedfor by the facts that thiosulfate is decomposed to sulfur andsulfite under the acidic condition at pH values below 4.5, andthat tetrathionate is reduced to thiosulfate enzymatically.In the oxidation of tetrathionate, oxygen uptake begins aftera lag phase, the duration of which depends on the concentrationsof cells and of tetrathionate. Cysteine is oxidized to cystine.The oxidation is strongly inhibited by metal-chelating agents.The cysteine oxidizing activity is, however, quite stable andis not lost by treating cells with organic solvents, sonic oscillation,by heating or lyophilization. ¹III=References (11). ²Partly supported by a grant from the Ministry of Education.     

STUDIES ON THE PATHWAY OF SULFIDE PRODUCTION IN A COPPER-ADAPTED YEAST

Metabolism of some sulfur-containing substances was studiedin a copper-resistant strain of yeast (R), its parent strain(P) and respiratory-deficient(RD) mutants from them. The resultsobtained are as follows:

1. Using sulfate, sulfite and thiosulfate as sulfur sources, Rproducedmore H₂S than P, and both of these had the activityhigher than their RD mutants. All of them produced a large amountof H₂S from cysteine, but only little from methionine, cysteinesulfinic acid and S-sulfocysteine.
2. From sulfite and thiosulfate,P and R produced more H₂S inaerobicthan in anaerobic condition.With sulfate and cysteine, however,H₂S production did not differunder those conditions.
3. In both P and R, the sulfate-to-sulfiteand sulfite-to-sulfidereactions were remarkably lowered byiron and zinc deficiencies.But the cysteine-to-sulfide reactionwas not affected by themetal-deficiencies.
4. H₂S productionfrom sulfate was remarkably depressed by highconcentrationsof pantothenate.
5. Rates of reaction steps on a plausible pathway from sulfatetosulfide and to organic sulfur compounds areestimated forthe strainsused. R is characterized by its largecapacity ofthe reaction step from sulfate to sulfite, and excessivesulfitethus formed is liberatedas sulfide not by the way ofcysteine.

¹Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka     

Sulfur requirement of Gonium (Volvocaceae)

The sulfur requirement of six strains of three species of Goniumhas been investigated. These strains can grow well with sulfide,sulfite, bisulfite, thiosulfate or sulfate in light and darkness.They are the first algae shown to utilize sulfide as a sulfursource. However, organic sulfur sources (methionine, cystine,cysteine, homocysteine, homocystine and taurine) were ineffectivefor growth of Gonium. (Received December 6, 1975; )     

PROMOTION OF ADVENTITIOUS ROOT FORMATION BY HELIANGINE AND ITS REMOVAL BY CYSTEINE

1. Heliangine at 10^–4M promoted the adventitious root formationin hypocotyls of cuttings taken from light-grown (1,900 lux)seedlings of Phaseolus mungo. The promotion was almost completelyreduced by simultaneously supplied 310^–4M cysteine or1.510^–4M cystine, but not suppressed by 310^–4Mof reduced glutathione, alanine or serine.
2. A 4 hr pretreatmentwith 310^–4M cysteine made Phaseoluscuttings less sensitiveto heliangine, but cysteine suppliedafter the treatment withheliangine brought about no effecton the action of heliangine.
3. Cysteine also removed the inhibiting effect of heliangineonthe indoleacetic acid-induced elongation of etiolated Avenacoleoptile sections.
4. In an aqueous solution heliangine formedan addition productwith cysteine, indicating that cysteinecan inactivate helianginewithout any biological processes.
5. On Phaseolus adventitious rooting, no effect was observedofp-chloromercuribenzoic acid, N-ethylmaleimide, 1,4-naphthoquinone,coumarin or penicillin. Reactivity toward sulfhydryl groupsalone does not qualify a substance to be a promotor of rootformation.
6. Maleic hydrazide at 10^–4M promoted root formation,butits effect was not removed by cysteine.

¹ Contribution No. 13 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo.     

Studies of sulfate utilization by algae 17. Reactions of the adenosine 5'-phosphosulfate (APS) sulfotransferase from Chlorella and studies of model reactions which explain the diversity of side products with thiols

Adenosine 5'-phosphosulfate sulfotransferase has been partiallypurified from Chlorella and is shown to catalyze the transferof the sulfate group of adenosine 5'-phosphosulfate to a varietyof thiol acceptors to form the corresponding organic thiosulfate.While the normal acceptor in the sulfate reducing pathway isthought to be a peptide carrier containing a thiol group theenzyme is very non-specific with respect to the thiols to whichit will transfer leading to a large number of side reactionswhich are possible when thiols are added to the system. Usingadenosine 5'-phosphosulfate and the enzyme, monothiols formsulfite and the organic thiosulfate of the thiol, with dithiolswhich readily form intramolecular disulfides, sulfite is theonly product, while with vicinal dithiols, sulfite and finallythiosulfate is formed. The -SO₃^– sulfur of the thiosulfateoriginates from adenosine 5'-phosphosulfate while the -S^–-sulfur is supplied by the vicinal dithiol. The same productscan be obtained using glutathione-S-sulfonate in place of adenosine5'-phosphosulfate and the enzyme, in a non-enzymatic reactionwith the same thiols. Thus it appears that the enzymatic reactioncatalyzes the transfer of the sulfate group of adenosine-5'-phosphosulfateto a thiol carrier or to any other thiol. When these other thiolsare present, however, sulfite, thiosulfate or organic thiosulfatesof the thiols are formed in non-enzymatic side reactions. Thetransferase from Chlorella is specific for adenosine 5'-phosphosulfateand will not catalyze the reaction with adenosine-3'-phosphate-5'-phosphosulfate. ¹Supported by Grants GB 4321, GB 40856X and BMS 73 00987 AO1from the National Science Foundation. ²Supported by a Gillette Graduate Fellowship. Portions of thispaper formed part of a dissertation presented to the graduatefaculty of Brandeis University in partial fulfillment of thePh.D. Degree. (Received June 30, 1976; )     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

Utilization of cystine by dermatophytes on glucose-peptone media

All the 16 strains of dermatophytes tested here metabolized cystine (3 mmol/L) in two glucose-peptone media with a different C: N ratio. Cystine was utilized as a sulfur source and, in addition, as a carbon and nitrogen source, in parallel with growth. Excess sulfur was excreted to the medium after its oxidation as inorganic sulfate and sulfite. In a physiologically alkaline medium the growth was fast and was accompanied by a pH increase and cystine was utilized intensively. Eleven species used up all cystine available. Sulfate was the main oxidation product, sulfite was produced at a low concentration, at the beginning of growth in particular. Only traces of thiol compounds (cysteine) were present in the medium. In a physiologically acid medium growth was soon limited by a decreased pH (below 5.0) but cystine continued to be utilized at an identical rate. All cystine was used up by 5 species. The tendency to produce sulfite in addition to sulfate further increased and sulfite was often the predominant product. Concentrations of thiol compounds were also substantially higher. Thus, dermatophytes can utilize cystine even under conditions that do not support good growth and increase the sulfite production.     

Influence of Glutathione (GSH) on Sulphate Influx, Xylem Loading and Exudation in Excised Tobacco Roots

In short-term experiments sulphate influx of excised tobaccoroots {Nicotiana tabacum L. var. 'Samsun') followed monophasicMichaelis-Menten kinetics with an approximate K_m of 12 ±4 µM and v_max of 657 ± 211 nmol g^–1 FW h^–1.An inhibition of sulphate influx, xylem loading and exudationof more than 70% was achieved with 01 mM GSH within 1 h. Cysteinewas two orders of magnitude more effective as an inhibitor thanGSH. An inhibition of more than 75% was already obtained with1.0µM cysteine. It may, therefore, be assumed that GSHis decomposed to yield cysteine concentrations that may inhibitsulphate influx, xylem loading and exudation. When BSO, a specificinhibitor of the initial step of GSH synthesis, was added, cysteine-mediatedinhibition on sulphate influx, xylem loading and exudation wasstrongly diminished. Apparently, GSH synthesis is required toobtain inhibition of these processes by cysteine. The physiologicalmechanisms that may cause the inhibition of sulphate influx,xylem loading and exudation by glutathione are discussed. Key words: Sulphate transport, Nicotiana, Solanaceae, glutathione, cysteine, buthionine sulphoximine     

Characteristics of Sulfite Transport by Chlorella vulgaris

The rate of short-term accumulation of [³⁵S]sulfite in Chlorellavulgaris cells was found to be strongly dependent on the pHof the medium. The rate increased with decreased pH, and theincrease in rate closely paralleled the increase in the concentrationof the un-ionized form of sulfite. When the pH of the mediumwas increased, fast accumulation ceased immediately. The rateof accumulation showed a strong temperature dependence, withan apparent temperature coefficient of 1.93 per 10°C rise,between 10 and 25°C. Because pK_a values of sulfite shiftwith temperature, the rates were corrected by dividing by theconcentration of the un-ionized form of sulfite present at therespective temperatures. The temperature coefficient was thenfound to decrease to 1.45. When cells which had been allowedto accumulate [³⁵S]sulfite for 20 min were transferred to amedium containing no sulfite, more than 50% of the accumulated[³⁵S] was released into the medium in 20 min. Our results arecompatible with a simple diffusion model of SO₂ transport intoChlorella cells. (Received September 26, 1996; Accepted January 20, 1997)     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

PRODUCTION OF HYDROGEN SULFIDE FROM SULFITE BY A COPPER-ADAPTED YEAST

Activity of hydrogen sulfide production from sulfite was studiedusing a copper-resistant yeast strain (R), its parent strain(P), and the culture of R in the medium without copper addition(R(0)). More hydrogen sulfide was produced under aerobic conditionthan under anaerobic condition. Sulfide producing activity wasin the order of R(0)>P>R under either condition. Stationaryphase cells produced more sulfide than logarithmic phase cellswhen cultured without copper, while the reverse was the casewith R, cultured in copper medium. Sulfide production was inhibitedby high concentrations of sulfite and by salicylaldoxime. Differencein the pathway from sulfite to sulfide was suggested betweenthe resistant strain (R and R(0)) and P in that the former wasmore sensitive to these inhibitors. ¹ Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka     

Donation of Electrons from Cytosolic Components to the Intersystem Chain in the Cyanobacterium Synechococcus sp. PCC 7002 as Determined by the Reduction of P700+

Cells, of Synechococcus sp. PCC 7002 showed a low oxidationlevel of P700 under a far-red light at 6 W m^–2 which inducednearly complete oxidation of P700 in spinach leaves, and a strongerfar-red light was required to observe the oxidation of P700.DCMU did not affect the level of P700⁺² but 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneinduced the oxidation of P700 under far-red light, indicatingthat the low oxidation level of P700 was due to the donationof electrons to P700⁺² from the cytosolic respiratory donorsthrough the intersystem chain at the plastoquinone pool. Theelectron transfer from the cytosolic donors to the intersystemchain was inhibited by HgCl₂ but not by antimycin A. The reductionof P700⁺ in Synechococcus cells, after illumination by strongfar-red light was mostly accounted for by the electron flowto the inter system chain from the respiratory donors (t     

Adenosine 5'-Phosphosulf ate Sulfotransferase from Euglena: Enzyme-Bound Intermediates

Adenosine 5'-phosphosulfate sulfotransferase (APSST) purifiedfrom Euglena gracilis Klebs var. bacillaris mutant W₁₀BSmL byammonium sulfate precipitation, Sephadex G-100 gel filtration,reactive blue agarose, reactive dye agarose and DEAE-cellulosecan be labeled by incubation with AP³⁵S and separated from smallradioactive compounds on Sephadex G-50. Most of the label isnot exchangeable with nonradioactive APS and therefore is notassociated with bound substrate. On non-inactivating SDS-PAGE,a radioactive band at the position of native APSST tetramershows APSST activity (measured as acid-volatile radioactivity).Labeled protein hydrolyzed with Pronase yields radioactive S-sulfocysteine,indicating that at least one cysteine residue of APSST acceptsa sulfo group from APS to form E-S-SO₃^–. A labeled lowmolecular weight compound can be separated from the proteinby paper electrophoresis or by treatment with acidic proteindenaturing reagents such as trifluoroacetic acid (TFA) or trichloroaceticacid (TCA). This labeled compound (perhaps the sulfo-carrier)behaves as a strong acid on paper electrophoresis and is stabilizedby iodoacetamide or acidic conditions but degrades to thiosulfate,sulfate and other compounds as the pH is raised. The radioactivityin APSST is exchangeable with sulfite or thiosulfate. AMP inhibitsAPSST in the formation of acid-volatile radioactivity by competingwith APS, but APA inhibits APSST activity uncompetitively. AK_m of 0.1 µM for APS and K_i of 0.1 mM for AMP and 0.6mM for APA are obtained when a saturating amount of dithiothreitol(DTT) is used as the thiol. A mechanism is proposed for theinitial reaction(s) catalyzed by APSST. ¹Present address: Boyce Thompson Institute for Plant Research,Tower Road, Ithaca, NY 14853, U.S.A.     

Studies on the metabolism of a sulfur-oxidizing bacterium IX. Electron transfer and the terminal oxidase system in sulfite oxidation of Thiobacillus thiooxidans

The nature of the electron transfer and terminal oxidase(s)in the sulfite-oxidizing system of Thiobacillus thiooxidnaswas studied in detail with various artificial electron donorsand inhibitors. Thionine, when reduced by ascorbate, was mosteffectively oxidized by whole cells and the particulate fractionof the various artificial electron donors. p-PD and TMPD werescarcely oxidized by either intact cells or the particulatefraction. The optimum pH of the thionine-oxidizing activity by the particulatefraction was 7.0 and that of the sulfite-oxidizing activitywas 6.8. The K_m values for thionine and sulfite were 7.6x10^–5Mand 1.6xl0^–4M, respectively. Sulfite oxidase activity in the particulate fraction was markedlyinhibited by amytal, rotenone, quinacrine-HGl and 2,4-DNP. HOQNOinhibited sulfite oxidase activity completely, but had no effecton thionine oxidase activity. Cyanide- and azide-insensitive respirations were present inthe particulate fraction. Thionine oxidase activity was inhibitedphoto-irreversibly with carbon monoxide, while sulfite oxidaseactivity showed photo-reversible carbon monooxide inhibition.The presence of two carbon monoxide-binding pigments was confirmedin the particulate fraction by a spectrophotometric study. (Received May 16, 1975; )     

Comparative studies on the effects of anions on ATPase and acid phosphatase activities from different sources

The effect of several anions on Mg²⁺-ATPase activity [EC 3.6.1.3 [EC] ]was studied in crude extracts of the following sources: twelvespecies of bacteria, one fungus, one yeast, six species of algae,five higher plant tissues, and two animal tissues. All the materialsexamined exhibited Mg²⁺-ATPase activity at pH 7.5. Except fora few species of bacteria, blue-green alga and etiolated seedlingsof a leguminous plant, Mg²⁺-ATPases of all the materials studiedwere stimulated two- to ten-fold by the addition of Group VIanions such as sulfite, selenite, and chromate. This stimulationseems to be a common characteristic of ATPases of most bacteria,chloroplasts, and mitochondria. On the other hand, the stimulation of acid phosphatase by sulfatewas not observed in any organism other than Thiobacillus thiooxidans. (Received July 27, 1977; )     

Differential modulation of voltage-dependent K+ currents in colonic smooth muscle by oxidants

The effect of oxidants on voltage-dependent K⁺ currents was examined in mouse colonic smooth muscle cells. Exposure to either chloramine-T (Ch-T), an agent known to oxidize both cysteine and methionine residues, or the colon-specific oxidant monochloramine (NH₂Cl) completely suppressed the transient outward K⁺ current (I_to) while simultaneously enhancing the sustained delayed rectifier K⁺ current (I_dr). In contrast, the cysteine-specific oxidants hydrogen peroxide (H₂O₂) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) exhibited partial and slow suppression of I_to by inducing a shift in channel availability of -18 mV without affecting I_dr. After enhancement by NH₂Cl or Ch-T, I_dr was sensitive to 10 mM tetraethylammonium but not to other K⁺ channel blockers, suggesting that it represented activation of the resting I_dr and not a separate K⁺ conductance. Extracellular dithiothreitol (DTT) partially reversed the effect of H₂O₂ and DTNB on I_to but not the actions of NH₂Cl and Ch-T on either I_dr or I_to. Dialysis of myocytes with GSH (5 mM) or DTT (5 mM) prevented suppression of I_to by H₂O₂ and DTNB but did not alter the effects of NH₂Cl or Ch-T on either I_dr or I_to. Ch-T and NH₂Cl completely blocked I_to generated by murine K_v4.1, 4.2, and 4.3 in Xenopus oocytes, an effect not reversible by intracellular DTT. In contrast, intracellular DTT reversed the effect of H₂O₂ and DTNB on the cloned channels. These results suggest that I_to is suppressed via modification of both methionine and cysteine residues, whereas enhancement of I_dr likely results from methionine oxidation alone. colon; colitis; redox; ion channel     

Effect of O2 Concentration on Dark H2 Oxidation in Whole Cells of a Marine Sulfur Photosynthetic Bacterium (Chromatium sp. Strain Miami PBS 1071)

Whole cells of photoanaerobically grown Chromatium sp. strainMiami PBS 1071, a marine purple sulfur bacterium, oxidized H₂in the dark through the oxyhydrogen reaction. Oxidation of H₂was measured by injecting either H₂ into an air-equilibratedcell suspension (microaerobic H₂ oxidation) or O₂ into an H₂/Ar-equilibratedcell suspension (microaerobic H₂ oxidation). Both types of H₂oxidation were strongly inhibited by azide (40 mM), indicatingthat the oxidation proceeds via a terminal oxidase system. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone(16 µM) inhibited aerobic H₂ oxidation by 49% but it acceleratedmicroacrobic H₂ oxidation. The sensitivity of H₂ oxidation torotenone was higher under aerobic conditions. The results indicatethat H₂ oxidation proceeds via two different pathways; one containsubiquinone and NAD, and the other does not. The contributionof each pathway depends on the O₂ partial pressure. ⁴ Present address: Institute of Oceanic Research and Development,Tokai University, Shimizu, Shizuoka 424, Japan. (Received May 24, 1985; Accepted August 29, 1985)