Acidithiobacillus

1
Acidithiobacillus
Acidithiobacillus ferrooxidans fixed on a grain
of pyrite(http//www2.brgm.fr/DMA/Chapitres/1Risq
ueEnviron/2Origine/Origine.html)
2
(No Transcript)
3
Acidithiobacillus phylogeny

• Proteobacteria
• Class III Gammaproteobacteria
• Order I Chromatiales (purple sulfur)
• Order II Acidithiobacillales
• Family I Acidithiobacillaceae
• Genus I Acidithiobacillus
• Order V Thiotrichales (filamentous sulfur)
• Order XIII Enterobacterales (E. coli)

4
Acidithiobacillus ferrooxidans

• Formerly known as Thiobacillus ferrooxidans
• Previously the Thiobacilli included most
  rod-shaped Gram- aerobic chemolithotrophs that
  oxidized sulfur-containing compounds.
• There was a classification revision in 2000
• sequenced rRNA genes of the Thiobacilli
• members of this genus fell into the ?, ?, and ?
  subgroups of the proteobacteria
• Thiobacillus is a ?-proteobacterium
• Acidithiobacillus is a ?-proteobacterium

5
Characteristics of Acidithiobacillus

• Acidophilic
• pH limits are 0.5 - 5.5
• Most species prefer pH 2 - 3
• Obligatorily aerobic
• All can use sulfur (S0) as electron donor
• Some can also oxidize
• sulfide (S2-)
• thiosulfate (S2O32-)
• Fe2 (only A. ferrooxidans)

6
Acidithiobacillus ferrooxidans

• Obligatorily autotrophic
• Use Rubisco Calvin cycle
• Make carboxysomes when CO2 is low
• Electron donors
• sulfur (S0)
• sulfide (S2-)
• thiosulfate (S2O32-)
• Fe2

7
Acidithiobacillus ferrooxidans

• Acidiphic
• pH optimum 2.5
• pH limits 1.3 - 4.5
• Oxidize sulfur compounds to H2SO4
• Found in acid mine drainage waters
• Optimum temperature 30-35C
• Can grow in range of 10 - 38C
• Can fix N2, but cannot reduce NO3-

8
(No Transcript)
9
Genome sequenced
10
Energetics of sulfur oxidation

• H2S 2 O2 ? H2SO4
• ?G -800 kJ/mol
• 2 H2S O2 ? 2 S0 2 H2O
• ?G -420 kJ/mol
• 2 S0 2 H2O 3 O2 ? 2 H2SO4
• ?G -1175 kJ/mol
• H2S2O3 H2O 2 O2 ? 2 H2SO4
• ?G -820 kJ/mol

11
(No Transcript)
12
(No Transcript)
13
(Taken from Rohrwerder Sand
)
14
(No Transcript)
15
(No Transcript)
16
(No Transcript)
17
Bioenergetics of iron oxidation

• Em of Fe(II)/Fe(III) 770 mV
• Em of Fe(II)/Fe(III) 650 mV at pH 2
• Em of O2/H2O 820 mV
• Not much energy to extract from iron oxidation.
• Bacteria must oxidize a LOT of iron to fix CO2.

18
Pathway of iron oxidation

• Iron(II)cyt c oxidoreductase
• Fe2 cyt c (Fe3) ? Fe3 cyt c (Fe2)
• Seems to be unstable in presence of cyt c (?).
• Multimer (10) of small subunit (6 kDa), each
  containing 1 Fe4S4 cluster.
• Iro protein (small subunit)
• Gene cloned and protein purified 50-55 residues
  (plus signal sequence)
• Similar to HiPIPs (high-potential FeS proteins)
  of purple sulfur bacteria
• Has Fe4S4 cluster is acid-stable

19
Model of structure of HiPIP
20
Pathway of iron oxidation

• There are several electron transfer cofactors in
  cytoplasm and inner membrane
• Rusticyanin
• Acid-stable Cu protein similar to plasticyanin
• Electrons are then transferred from cyt c to
  rusticyanin spontaneous or catalyzed?
• Cyt c552(s) standard cyt c
• Membrane-associated Cyt c
• Cyt c552(m)
• Cyt c550(m)

21
Pathway of iron oxidation

• Acidithiobacillus contains a cyt c oxidase
• Fe2 cyt c (Fe3) ? Fe3 cyt c (Fe2)
• It is the aa3 type
• Only expressed when growing on Fe2
• Like most cyt ox enzymes, except acid-stable
• Can accept e- from
• Rusticyanin
• Soluble cyt c (s)
• Membrane-associated Cyt c552(m) Cyt c550(m)

22
(No Transcript)
23
Process of iron oxidation
Cyt c (Fe3)
?
Fe2
Cyt c (Fe2)
Rc (Cu1)
Fe3
Cyt oxidase
O2 4 H 2 H2O
24
Proton pumping vs. proton consumption?

• Since A. ferrooxidans grows at pH 2-4, there is
  already a significant pH gradient across its
  inner membrane.
• All it has to do is consume the protons
  transported across by ATP synthase.
• Cyt oxs job
• Some of the gradient can also be used for reverse
  electron flow.

25
Reverse electron transfer

• How do you reduce NAD(P)??
• Electrons from Fe(II) dont go to quinone pool
• Cannot just drive NADH DH backwards
• Solution drive cyt bc1 backwards too
• The genome encodes 2 different cyt bc1 complexes
• The one expressed in S-grown cells works in
  forward direction QH2 2 cyt c(Fe3) ? Q
  2 cyt c(Fe2)
• The one expressed in Fe(II)-grown cells only
  works in reverse 2 cyt c(Fe2) Q ? 2 cyt
  c(Fe3) QH2

26
(No Transcript)
27
S-grown cells

• Also express alternate terminal oxidases
• Quinol oxidases 2 QH2 O2 ? 2 Q 2 H2O
• Cyt bd
• Cyt bo3
• Cyt oxidases 4 Cyt cred O2 4 H ? 4 Cyt cox
  2 H2O
• Cyt aa3 (also in Fe-grown cells)
• Cyt ba3
• Reason perhaps to scavenge O2 to allow
  nitrogenase to function

28
(No Transcript)
29
Oxidation of S-containing amino acids

• Cysteine (all S released from Cys)
• Cysteine dioxygenase uses 2 NADH to activate 2 O2
  molecules to oxidize the thiol to a sulfinic
  acid.Cys 2 O2 2 NADH 2 H ?
  Cysteinesulfinate 2 NAD 2 H2O(Cys is not
  immediately transaminated, as the product of this
  first enzyme is a branchpoint metabolite.
  Cysteinesulfinate can be converted to the bile
  acid taurine, by decarboxylation and oxidation.)
• The sulfinate is transaminated to ?-sulfinyl
  pyruvate.
• Release of SO2 (desulfuration) gives pyruvate.
• The SO2 can spontaneously react with OH- to give
  bisulfite (HSO3), which can deprotonate to
  sulfite. (Think of CO2 reacting to form
  bicarbonate and carbonate)

30
Sulfite oxidoreductase

• Sulfite is detoxified by all organisms.
• Eukaryotes contain the enzyme sulfiteacceptor
  oxidoreductase to oxidize sulfite to
  sulfateSO32 2 cyt cred ? SO42 2 cyt cox
• The enzyme is mitochondrial and localized to the
  inter-membrane space (IMS, analogous to bacterial
  periplasm).
• In (almost) all cases examined, the electron
  acceptor is cytochrome c (also in periplasm or
  IMS).

31
Mechanism of Sulfite Oxidoreductase

• All SORs have a form of molybdopterin in the
  active site (also in nitrate reductase)
• The Mo cycles between 4, 5, 6 oxidation states
  during catalytic mechanism.

32
SOR structure
33
SOR general mechanism
34
SOR from Thiobacillus novellus

• Heterodimeric enzyme
• SorA MoPterin
• similar to eukaryotic SOR in Mo cofactor domain
• Lacks heme b domain
• SorB cyt c
• unrelated to other cyt c
• Kinetic analysis
• Ping-pong mechanism
• 2 distinct sites
• Km(SO32) 30 µM, Km(cyt c550) 4 µM

Kappler et al. (2000), JBC 27513202
35
SOR from Acidithiobacillus ferrooxidans

• New type of SOR found
• Did not reduce cyt c or ferricyanide
• Did reduce Fe3 in presence of phenanthroline
  (chelator)
• Potential problems
• Only used crude membrane prep as enzyme
• pH optimum 6
• Never followed up on
• Suggests that the organism stores reducing
  equivalents as Fe2, which it later oxidizes.

Sugio et al. (1988), Appl. Env. Microbiol.
54153
36
SOR from Acidithiobacillus ferrooxidans
Sugio et al. (1988), Appl. Env. Microbiol.
54153