A study of electron transport in Propionibacterium shermanii : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

A STUDY OF ELECTRON TRANSPORT IN PROPIONIBACTERIUM

SHERt-1AN I I

A THES I S

pre sented i n part i al fulf ilment of the requi rements for the degree of Doctor of Philosophy in B iochemi stry

at

Mas s ey Univers tiy

RoDERICK VINCENT AsMUNDSON 1982

, ; f! •. ^. c. 'i

ACKNOWLEDGEMENTS

Thanks are due to many people who he lped in various way s in the produc tion of thes i s . In parti cular ,

thanks are due to :

Dr G . G . Pritch ard , who provided much h e lp in a gent lemanly , even tempe red manner .

Nrs P . Lyttleton , who performed mo st o f the electron mi croscopy .

Mr N . Foote ( machin i s t ) , Mr S . Oliver ( gl as sblower ) and - Mr P . Mintoft ( e lectri cian ) who provided e f f i c i ent and

c reative s ervice on s ome instruments used in thi s work . Mr D . Fenemore and D r. I . -Andrews , who provided a method

for c arbohydrate ana ly s i s .

Barbara Asmundson , who to l erated t he extra time requi red to p erform the re s earch as we l l as t he poor humour which accompanied portions o f the work .

ABSTRAC'r

Propionib acteria po s se s s a m�mbrane-bound e le ctron transport system coupl ing ox idat ion o f several s ubstrate s to reduct ion o f fumarate or oxygen . Publ ished work has ind icated that the e lectron t ransport system con s i s t s o f the fol lowing : deh

y

drogenases for NADH , lactate , glycerol pho s phate and succinate; menaquinone ( I I , I I I -te trahydro­

menaquinone - 9 ) ; cytochrome s b , a , d and poss ibly o ; terminal oxidase ( s ) and fumarate reductase . Menaquinone has been demons trated to be an obli gate component o f e lec tron transpo rt to fumarate but the re i s d i s agreement in the l i te rature c oncerning th e role of the b-cytochrome components in the e le c tron transport pathv1ays . In the pre s ent s t udy , the e f fect of a nuwber of inh ib i tors on

electron tran�port from NADH , D- and L-lactate and succ inate to oxygen and to fumarate was investigated with the aim o f e lucidating the relationship b etween the aerobi c and anaerobi c pathways for oxidat ion o f these sub strates and the role o f the cytochr�me component s in these sys tems .

Membranes were p re pa red by b reaking l ac tate -grown c e l l s o f P. shermanii ATCC 9 6 1 4 i n a Hughes p re s s and

separating memb rane s from solub le components by di ffere ntial centr i f ugation w i th a final centri fugation ov�r a s tep\..; i se suc rose gradien t .

Spectrophotometric analy s i s o f pyridine hemochromogen derivative s showed the p re sence o f s igni fican t quan t i t i e s o f cytochromes b and d , but d i d no t de tect the p re sence o f cytochrome a or c . The cytochrome s were a l so characte ri zed

by redox ti trimetry at room temperature and by analy s i s o f

/

low temperature s pec tra o f redox-·poi sed· membranes . The se expe r iments ind i c ated the presence o f four b -type cyto chrome s with a-absorption pe aks ( s ubs crip t ) and approximate mid-

point redox poten t i a l s at pH fo l lows : b5 6 2 _ 3 ( +1 2 0 mV) ;

7 {bracketted f i gure s ) as

HP ^· LP

b5 5 6 _ 7 ( +90 mV ) ; b5 5 6 _ 7 ( -2 0 mV ) ; b5 5 3 _4 (,....2 0 mV ) . A midpo int potenti a l a t pH 7 for cytoch rome d of approximat e ly +150 mV was determined from room temperature t itrations .

O f seve ral inh ib i tors surveyed for e ffe c t s on e l e ctron transport activi t ie s of memb rane s , pCMB , d i cumarol , UV l ight and cyanide were s e lected for detailed s tudy . Dicumarol wa s particu l ar ly e f fective as an inhibitor o f L-lactate dependent activitie s . L-l actate -dependent reduct ion of oxygen , D IC P I P and Fe ( CN ) 6 was inh ibited by a concentrat i on o f dicuma ro l 1 0 3 times le s s than tha t required f o r comp arab l e inh ib i tion o f the corre sponding D - l actate and NADH dependent activitie s . Thi s indic ate s either tha t the L-l actate dehydrogenas e has d i s t in ctive

?icumarol - sensitive compon ents or that i t i s inte grated

in the membrane in a manner which enhance s i t s acce s s ib i l i ty to dicumarol . The f ac t that the D-l actate -coupled

activitie s are ve ry s table whi le the L- lactate dependent a ctivi t i e s are unstabl e also indi cate s th at t he two l ac tate dehydrogenase s ystems h ave d i f ferent propert i e s .

Ultra�iolet l i ght i rradiation caused a rapid inactivation o f NADH- and D-lactate - fumarate oxidoreductases but a

s l<;:>.wer inac tivat ion o f NADH and D- lac tate ox·idase s . Th i s was inte rp reted t o i ndic ate that e lectron t ransport to

fumarate has an ab so lute requi rement for menaquinone wh i le a porti on o f the ox idase activi ty did not requi re

menaquinone .

All e le ctron transport a ctivitie s we re inhibited by

-4 .

pCMB at a concentra t i on around 1 0 mo les per g ram protein but NADH-dep endent red uction o f oxygen , Fe (CN) 6 and DICP IP was inhibi ted to a maximum of only 5 0 % .

Th e pCMB titrat i on curve s for inhibition o f NADH-

�

dependent activitie s and the s igni ficantly h i gher reduction o f cytochrome b by NADH than by D- o r L - l actate i n the

presence of i nhibi tory concentrations o f pCr.ffi sugge s t the presence of two independent dehydrogenases for NADH .

However the c ompl ete i nh ibition o f NADH -fumarate oxidoreduc - t a s e b y pCMB s ugge sts that only the pCMB -sens i t i ve dehydro- genas e is i nvo lved in ele ctron transport to fumarate .

Cyanide inhibi ted · D- and L-lactate - and NADH- dependent oxygen consump tion w ith an r5 0 of about 2 mM. The

r e l at ive ly l ow sens i t i vi ty to cyani de sugge s t s th at cytochrome d is the m a j or terminal oxi das e . Thi s i s consi s te nt with th e evidence f rom d i f ference spec tra o f cyan i de -inhibi ted aerob i c s teady s tate membrane s indicating that cyanide bind s to cyto chrome d . and inhi b i t s oxidation o f cytoch rome b . Al though a eo-bindi ng cytochrome b i s pre sent , the low cyan i de s:ens i ti vi ty and abs e nce o f CO

inhibition o f oxygen uptake s ugge s t that an o-type oxidase does no t contribute s i gni fic antly to oxygen uptak e .

The e f fect o f inhib i tors on reduct i on o f Fe ( CN ) r and

0

DICPIP by a l l four s ub strates was also systematical ly in.ve stigate d . The re sults obtained from the s e studie s

suggest multiple site s o f interact ion o f the se a c ceptors

with the etectron tran sport pathways, the points o f inte r action depending on both the nature o f the a cceptor and o£ the reducing sub strate .

Add i tion o f fumarate c aused a dec rease o f 2 0 - 3 0 % in

the rate of oxygen con sumpt ion and in total oxygen con sumption at concentrations between 0 and 1 mM. No furthe r

i nhibition occurred with increas ing fumarate conc entrations up to 1 0 mM, but concentrations above 1 0 mM inhibited first the rate o f oxy gen c onsumption and then the tot a l oxygen con sump tion unt i l both we re comp l ete ly inhibi ted at 40 mM fumarate� I nhibiJcion at low concentrations o f fumarat.e i s probably due to dive r s ion o f a portion of t he e lectron f l ow f rom oxy gen to reduction o f fumar ate . Thi s

competi tion b etween oxy gen and fumarate for e lec trons indicate s th at the pathway s to these two acceptors share common i n i tial s tep s . ·

Comparison of cytochrome di fference spectra o f membranes i n the aerobic and anaerobic s tate in the pre s ence and

absence o f .··,in .hibi tors indicated that UV , dicumarol and pCMB inhibi ted e lec tron trans port at s ite s on the reduc ing s ite of the cytoch rome b components . Concentrat ions o f pCMB whi c h comp l ete ly inhibited fumarate reduct i on appeared

to leave cytochrome b5 5 3_ 4 large ly in the reduced s ta te in the anaerobi c steady- s tate , but not in the ae rob i c steady s tate suggesting that thi s cytochrome is not a component of e le ctron transport to fumarate . The other low potenti a l b type cytochrome b5 5 6 _ 7 was oxidi zed by fumarate i n the LP

pre sence o f pCMB , s ugges ting that i t may be l o cated

specifi c a l ly on the anaerobi c path to fumar ate . Since fumarate oxidized at l eust part of the cytoch rome b in UV- i rradiated me�bran es , a p revious· s cheme for e lectron

transport in thi s organism in which cytochrome b can only be oxidi zed by fumarate via menaquinone was not suppor ted by the p re sent s tudy .

Spectra o f memb rane s in the aerobic s te ady s tate indicate d a greater degree of reduction of cytochrome b56 2--3 \vhen s uc cinate was re ducing s ubstrate than when D-

o r L-la ctate or NADH was reducing s ubstrate . Thi s s ugge s t s that the re w a s a c l o s e re lation ship between succ inate

dehydrogenase and cytochrome b5 6 2 _ 3 . S ince s u ccinate oxida s e activity wa s low de spi te the presence o f high succin a te dehydrogenase activity , i t i s improbab le that b5 6 2 _ 3 is part o f an oxidas e .

The p re s�nt s tudy did no t reso lve the que s ti on as to whether the re was a sin g l e succinate dehydrogenase which

fun ctions in vivo as the fumarate re ductase . Mo s t evidence is c on s i stent with there b e1ng only orie enzyme sys tem but the f inding of two d i stinct K ( .

t ) va lues for

· m succ1na e

succ inate-DI CP IP and succinate -Fe ( CN ) 6 oxidoreductase may indicate the pre se nc e o f two d i stinct s ys tems .

TABLE OF CONTENTS

Page No . 1 : INTRODUCT ION

1 . 1 : Some General Ch aracte r i s t i c s of B ac te ri a l

E l ectron T ransport 2

1 . 2 : Th e Electron Transport Sy stem of Propioni-

bacteria 1 1

1 . 2 . 1 : E l e ctron transport ac tivities and c omponents in memb r anes from

propioniba cteria 1 3

1 . 2 . 2 : Organ i z ation o f the electron

t ransport sys tem in propionibacteria 1 7 1 . 3 : Aims o f the Present S tudy 2 4

2 : METHODS AND MATERIALS 2 . 1 :

2 . 2 : 2 . 3 : 2 . 4 :

Culture Methods

Preparation of Memb ranes E le ctron Micros copy

Ana lyti c al Methods

28 2 9 3 2

2 . 4 . 1 : P ro te in determination 3 3

2 . 4 . 2 : Lipids 3 3

2 . 4: 3 : Total carbohydrate s 3 3

2 . 4 . 4 : Gas chromatography 3 4

2 . 4 . 5 : Pyrid ine hemochromogen derivative s

o f cytochromes 3 5

2. 5 : De termination of E l ectron T r ansport

Activi t i es 3 5

2 . 5 .1 : 2 . 5 . 2 : 2 . 5 . 3 :

De te rfuination o f dehydrogenase activitie s

Fumarate reductase a ctivity Oxygen oxidoreduc tase act ivity

3 5 3 8 4 1 2 . 6 : Spectrophotometric Examination of Cytochrome 4 1 2 . 6 . 1 : Low temperature spectrophotomet ry 4 1

2 . 6 . 2 : Redox ti trations 4 3·

2 . 6 . 2 . 1 : ^. Room temperature redox t itration s

2 . 6 . 2 . 2 : Low temp erature redox t i trations

: . 2 . 7 : Sourc e s o f Ch emi cal s

4 3 4 5 4 6

P age No . 3 . RESULTS

3 . 1 : Ch arac te ri s tics of Memb rane Partic l es 4 9.

3 . 2

3 . 1 . 1 : Inve s t i gati on of th e membrane f ra c t ion by e lec tron microscopy and dens i t

�

^_

gradient cent ri fugation ^· 4 9 3 . 1 . 2 : E l ectron tran sport ac tiviti es o f

P. s he rmani i membrane prepara t ion s 6 4 3 . 1 . 2 . 1 : Spe c i f i c activities o f

oxygen and fum arate

oxidoreductase s 6 4

3 . 1 . 2 . 2 : Spe c i f i c activi te s o f Fe ( CN) 6 and DICPI P oxidoreductases

3 . 1 . 3 : Pro tein , carbohydrate and l i p i d conte nt o f membrane s

3 . 1 . 4 : Quinone con te nt o f membranes 3 . 1 . 5 : Cytochrome content o f memb rane s

3 . 1 . 5 . 1 : Gene ral characteri z a t ion o f 6 6

7 4 7 9 8 0

cy tochrome s 8 0

3 . 1 . 5 . 2 : Room tempe rature redox

titrations 9 2

3 . 1 . 5 . 3 : Low temperature spectra of

redox poi s ed memb rane s 9 8 3 . 1 . 6 : Su� ary o f c onclus ions from membr ane

characte r i z ation s tud ies 1 1 0 E f fects o f Inhibitors on Elec tron T ransport

Ac t ivities and·Reduct i on Level of the Cy tochromes i n Membranes o f P. sh e rma n i i 3 . 2 . 1 : Introduction to principles and

proce·dures used

3 . 2 . 1 . 1 : General de s cription o f experimental pro cedure 3 . 2 . 1 . 2 : Spectra o fvmembranes i n

1 1 3

1 1 3

sub s trate reduced and 1 2 1 s te ady s tates

3 . 2 . 2 : E f fe cts o f UV irradiat ion and n-

pen tane extraction 1 2 8

3 . 2 . 2 . 1 : E f fects o f UV i rradi ation on e lectron transport

activiti res 1 2 9

3 . 2 . 2 . 2 : E f fe c ts o f UV l i ght on reduction and oxid a t ion o f

_ cytochromes 1 3 4

3. 2. 2 . 3 : E f fects o f solven t extraction on ele ctron

transport activi t i e s 1 4 1 3 . 2 . 2 . 4 : Conclus i on s from UV

i rradi� t ion and n-pentane

extraction exper iments 1 4 6 3 . 2 . 3 : E f fe ct s o f di cumaro l on e l e c tron

tran sport: 1 5 1

3 . 2 . 3 . 1 : Dicumaro l inh ibi tion o f .

e le c t ron t ran sport , acti vi ti es

3 . 2 . 3 . 2 : E ffe cts o f di cumaro l on

1 5 2

cytoch rome reduction 1 5 3 3 . 2 . 3 . 3 : Conc lusions from

exper imen ts using di cumarol 1 5 7 as an inh{b i tor

3 . 2 . 4 : Inhibi ting e f fe ct s o f pCMB on e l e c t ron

transport proce s s e s 16 5

3 . 2 . 4 .1 : Inh ibi t ion of e l ectron

transport activi t i e s 16 5 3 . 2 . 4 . 2 : E f fects o f pCMB on reduc-

t ion leve l o f cytoch rome s 1 6 7 3 . 2 . 4 . 3 : Conclusions from the s tudy

o f pCMB inh ibi t ion 1 7 6 3 . 2 . 5 : I nhibi tory e f fects o f HOQNO on

e l ec t ron trans port p roce s s e s and 181 cytoch rome reduction

3 . 2 . 5 . 1 : Inhib i tion of e l e c t ron

t ransport activitie s 18 2 3 . 2 . 5. 2 : E f fects o f HOQNO on

cy tochrome reduct ion 1 8 5 3 . 2 . 5 . 3 : Conc lus ions from studies

with HOQNO 1 86

3 . 2 . 6 : E f fe ct s o f cyanide on electron tran sport

3 . 2 . 6 . 1 : E f fe cts o f cyani de on e l e ctron t ran sport

189

activit i e s 190

3 . 2 . 6 . 2 : E f fe c t o f cyan ide on stoi chiome try of oxygen

consumption 1 92

3 . 2 . 6 . 3 : E ffe cts o f cyani de on

cytochrome reduct ion leve l s 2 0 2 3 . 2 . 6 . 4 : Con clus ions from

experiments u s in g cyani de 2 0 8

3 . 2 . 7 : E ffects of fumarate

3 . 2 . 7 . 1 : E ffe c t s o f fuma rate on

Page

·No . 2 1 0 oxygen consumption 2 1 1 3 . 2 . 7 . 2 : E ffec t o f cyanide on

fumarate competi t ion 2 1 3

3 . 2 . 8 :

4 . D I SCU SS ION

3 . 2 . 7 . 3 : Low temper atu re spec tra o f memb r anes i n the ae rob i c s te ady s tate w ith £umarate 3 . 2 . 7 . 4 : Con clu s ions from th e

inve s ti gation of fumarate competition vli th oxygen consumption

Other inhibi to r s o f e lectron t ran sport

2 2 2

2 2 7

2 2 7

2 2 9 4 . 1 : An ae rob i c E l e ct ron Tran s port to Fumarate 2 3 0 4 . 2 : Th e Ae rob i c E l ectron Transport Pathway 2 3 8

4 . 2 . 1 : D i fference b etween reduc ing sub strate s in e l e c tron tran s po rt to oxygen 2 3 9 4 . 2 . 2 : The nature o f the te rminal oxidase 24 1 4 . 3 : The P re sence o f S teps Common to Both

Aerob ic and . Anaerobi c P athway s 2 4 3 4 . 4: I nt e ract ion s o f DICP IP and Fe ( CN ) 6 w i th the

E l ectron Transport Sys te m 2 4 5

4 . 4 . 1 : D - l actate 2 4 6

4 . 4 . 2 : L - l actate 4 . 4 . 3 : NADH

4 . 4 . 4 : Suc c in a te

4 . 5 : D i f fe rences be tween Pathways Oxidi zing D i f fe rent Donor s

4 . 5 . 1 : Di ffe rence s b etween pathways

2 4 8 2 4 9 2 5 0

2 5 1

oxid i z ing D- lact ate and L-l actate 2 5 2 4 . 5 . 2 : Di f fe rences b etween pathways

oxidi z ing NADH and D-lactate 2 5 3 4 . 5 . 3 : D i s tinc tive fe a ture s of s uccin a te

depende nt e l e c t ron t ran s port

a ct ivi ties 2 5 5

4 . 6 : The Rol e o f Cytoch romes i n E lectron Tran sport 2 6 0

4. 7 : Conc lus ion 2 6 4

L I S T OF F I GURES P age No .

1 . 1: Ae robi c Electron Tran sport 5

1 . 2: B acte r i a l Quinone s 7

1 . 3: La ctate Metabol i sm in P rop ionibacteria 1 2

1. 4: An ae rob i c Ele ctron Transport 2 3

3 . 1 . 1 . 1: D- l actate - D I CPIP Oxido reductase and

Total Prote i n in Fract ions from 5 7

3 . 1 . 2 . 1:

3 . 1 . 3 .1:

3 . 1 . 4 . 1:

3 . 1 . 5 . 1:

3 .1 . 5 . 2:

3 . 1 . 5 . 3:

3 . 1 . 5 . 4:

3 . 1 . 5 . 5:

3 . 1 . 5 . 6:

3 . 1 . 5 . 7:

3 . 1 . 5 . 8:

3 .1 . 6 . 1:

3 . 2 . 1 . 1:

3 . 2 . 1 . 2:

3 . 2 . 2 .1:

3 . 2 . 2 . 2:

Den s i ty Gradient Centrit.Lt1ation o f Memb rane s P repared f rom P . s he rman i i Li neweaver Burke Plots o f succinate ­ Fe ( CN ) 6 and s uccin ate-DICPIP

oxid'Jreductases

Gas - l iquid Ch romatography Tracing of Ace ty l ated Monosacch aride s

U V Ab sorption Sp ectrum o f the Quinone Extra cted f rom Mewbranes o f

P . s h e rmani i

Roont Temperature and Low Temperature Spe ctra o f Di th ioni te and Sub s t rate Reduced Membrane s

Carbon Monoxide Di ffe rence Spectra o f Membranes

Spe ctrum o f Pyri dine Hemochromo gens Room Temperat ure Spectr a of Redox Poi sed Membranes

Room Temp erature Redox Ti tra tions o f Cytoch rome s b and d

Low Temperatu re Spe ctra o f Redox Poi sed Membr anes

Low Temperat ure Spectra o f Redox Poi sed Memb ranes

Low Temperat ure Spectra of Redox Poi sed Memb r anes

E le ct ron Transport Components o f P. s he rma n i i Memb rane s ⁰

Dua l W ave leng th Spectrophotometer Trac i n g of Inh ib i ted and Uninh ibi ted Cytoch rome b Reduction

Spec tra o f Membrane s in Uninh i b i ted Sub s trate Reduced and S te ady States UV Inactivation o f E le ctron T r anspo rt Act i vi tie s

Effect s o f UV I rradi at ion on Reduction leve ls of Cytochron�s

7 2

7 7 81

84

8 8 9 0

� 3 9 5 1 0 2

1 0 5

1 0 7 1 1 2 11 5

1 2 3 1 30

1 36

3 . 2 . 3 . 1 :

3 . 2 . 3 . 2 :

3 . 2 . 4 . 1 : 3 . 2 . 4 . 2 : 3 . 2 . 5 . 1 :

Dicumarol I nhibition o f E le c t ron Transport Activiti e s -o f Memb rane Ve s i cle s

E f fe ct o f dicumarol on Subs trate Redu ced and S teady State Low Temperature Sp ectra o f Memb rane Ve s i c l e s

pCMB Inhibi tion o f Ox idoreduc tas e s P re sent in M emb rane s

•

E ffe cts o f pCMB on Reduction Le ve l o f Cyto chromes in Memb ranes

E f fe cts o f HOQNO on E l ectron Transporb Page

No.

1 5 4

15 8

16 8

1 7 1

Activi t ie s 1 8 3

3 . 2 . 5 . 2 :

3 . 2 . 6 . 1 : 3 . 2 . 6 . 2 : 3 . 2 . 7 . 1 : 3 . 2 . 7 . 2 : 3 . 2 . 7 . 3 : 3 . 2 . 7 . 4 : 3 . 2 . 7 . 5 :

E f fe cts o f HOQNO on Cy tochrome Re duction

E f fe cts o f Cyan ide on E lectron Transport Activi t ie s

E f fe c t s o f Cyanide on Cytoch rome Redu c ti on

Fumarate Inhibi t ion o f Oxyge n Con s umpt ion

Appa ren t Km (fum) for Benzyl Viologen­

Fumarate ox1aoreductas e . _.

K. (f ) for Succin ate ( +PMS ) -D I CP I P

0

�

^1a

8P

^{eductas e ·}

E f fe ct o f Cyanfde on Fumarate Compe ti.t ion with Oxygen

E ffe ct of Fumarate on the Ae rob ic S te ady S tate Reduction Leve l o f Cy tochromes in the P re sen ce and Ab sence o f 10 mM Cyan ide

4 . 1 : Tentative Scheme for E l ectron T ransport i n P . s he rman i i

1 8 7

1 9 4

2 0 4

2 1 4

2 1 6

2 16 2 19

2 2 4

2 6 6

L I ST O F TABLES

1 . 1 : Cytochromes o f the b type Pre sent in E. c o l i 9 1 . 2 : Speci fic Acti vi t ie s o f Memb rane Bound E le c tron

Trans port Rea c t i on s in p ropionib acteri a 1 4 2 . 4 . 5 . 1 : Ab sorption Characte ri s tics o f A lkal ine

Pyridin e Hemochromogens 36

2 . 6 . 2 . 1 : Redox Mediators 4 4

3 . 1 . 1 . 1 : Comparis on o f Recovery o f E le c t ron

Trans port Acti vi tie s in Memb rane s 5 3 Prepared W i th and W i thout Mg++ in the

Isola tion Buf fe r

3 . 1 . 1 . 2 : Sucro s e De nsity Gradient Fract ions

of P. she rma n i i Membrane s : P ro te in , 5 9 Spe ci fi c Ac t i vities and Spec i fi c

Gravi ty o f Fractions

3 . 1 . 1 . 3 : Parti cle Si ze s from Th ree Di fferent

Den s i ty Gradient Fractions 6 0 3 . 1 . 2 . 1 : Speci f i c Ac tivit ie s o f Oxygen and

Fumarate Oxidoreductase s in Membrane s

from P. s he rman i i 6 5

3 . 1 . 2 . 2 : Spe ci fi c Act i vi t i es o f Fe ( CN ) 6

Reducta s e s 6 6

3 . 1 . 2 . 3 : Spe ci f i c Activi tie s o f DICP I P Reductas e s 6 7 3 . 1 . 2 . 4� Effe ct s o f Oxygen on Fe ( CN ) 6 and DICP I P_

Reductases 6 8

3 . 1 . 2 . 5 : Effe c t s o f PMS on Fe ( CN ) 6 and DICPIP

Reductase s 6 8

3 . 1 . 2 . 6 : Appare n t ·Km Va lues f o r E le ct ron Donors

of Some Ox1dore ductase Sys tems 7 0 3 . 1 . 2 . 7 : Apparent Succ inate B inding Con stants for 7 1

Succinate -Fe ( CN ) 6 and Succin ate - D I CP I P Oxi do re ductase Sy stems

3 . 1 . 3 . 1 : Pro tein Analy s i s o f Membranes

3 . 1 . 3 . 2 : Carbohydrate and LipidvConten t o f Memb rane s

3 . 1 . 5 . 1 : Absorp tion Max ima and Approximate H i d-

7 5

7 6 point Potent i a l s o f Cytoch romes o f 1 1 0 P. s herma ni i

3 . 2 . 2 . 2 : Effe c t s o f n-pentane Extraction o f

Memb rane s on Rate o f Oxygen Con s umption 1 4 2 3 . 2 . 2 . 3 : E f fe c t s o f n-pentane Extraction o f

Memb ranes on DICP I P ·Redu ctase Activi ty 1 4 3 3 . 2 . 2 . 4 : The E ffec t o f n-pentane Ext raction o f

Membrane s cri Fumarate Redu c tase 1 4 4 Activi ty

3.2.2.4 : The e ffe ct o f Quinone Readdi tion to

Page -No .

N-pentane Extracted Membrane s 1 4 5 3 . 2.4 .1 : pCMB pi 5 o/E Values for D-lactate

Oxidoredu ctase s 16 7

3.2.5.1 : The E f fe c t o f Oxygen on HOQNO

S timulation 1 8 5

3.2.6.1 : E ffect o f Cy ani de on Stoichiometry o f

Oxygen Consumed to Substrate Oxidi zed 19 3 by Memb rane Parti c l e s of P. s he rma n i i

3.2.6 .2 : Cyani de I nh i b i tion o f Catal ase 2 0 0 3.2.6.3 : Production o f H2o2 in the P re sence o f

5 0 mM Cy anide 2 0 1

3.2.7. 1 : Compe ti tion o f Fumarate wi th

DICPIP Reductases 2 1 3

4 .4 .1.1 : Spe c i f i c Activi i tes of D- lactate

Oxidoreductas e s ^· 24 6

LIST OF PLATE S

3.1�1 . 1 : E lectron Mi crograph o f Membrane s Prepared

P age No:-

in the P resence o f Mg++ 5 0

3 . 1.1 . 2 : E le ct ron Mi crographs o f Ne gative S ta ined

Ce l l s o f P. s he rma n i i 5 4 3 . 1 . 1 . 3 : E le ctron Microgiaphs o f Pooled F ract ions

f rom Dens i ty Gradient Centri fugation 62

a , b , c or d CN

DIC DICPIP Fe ( CN ) 6 fpD fpFR fpL fpN fps HOQNO MQ NOQNO pCMB PMS uv

ABBREVIAT I ONS

Cytochromes a , b , c or d Cyani de

Dicumarol

2 , 6 -d i chloropheno lindopheno l potass ium ferricyanide

D-l ac tate dehydrogena s e Fumarate reductase

L-l actate dehy drogenase NADH dehydroge nas e

S u cc inate dehydrogenas e

N-heptyl-4 -hydroxyquinol ine-N-oxide Menaquinone

n-Nony l-4 -hydroxyquinoline�N-oxide p-chl0rome rcuribenzoic acid

Phena z ine rnethos ul fate U ltraviole t l i ght