Download H 2

Geobiology Week 3
How do microbes garner energy and carbon?
Review of redox couples, reaction potential and free energy yields
Hydrogen as an energy currency for subsurface microbes.
Acknowledgements: Tori Hoehler
Redox structure of modern microbial ecosystems
Deep biosphere as an analogue of Early Earth Ecosystems O2 as
a driver of biological innovation
Readings : Brock Biology of Microorganisms. Hoehler et al., 1998.Thermodynamic
control on hydrogen concentration in anoxic sediments Geochim. Cosmochim.
Acta 62: 1745-1756. Hoehler TM, et al., 2002. Comparative ecology of H2 cycling
in sedimentary and phototrophic ecosystems Antonie von Leeuwenhoek 81: 575
582. Hoehler et al., 2001. Apparent minimum free energy requirements for
methanogenic Archaea and Sulfate reducing bacteria in an anoxic marine
sediment. FEMS Microbial Ecol. 38; 33-41.
Microbiology
Ecology
Biogeochemistry
A staggering number of organism-organism and organismenvironment interactions underlie global biogeochemistry
These can be studied at vastly different spatial and time scales
PRESS RELEASE
Date Released: Thursday, February 21, 2002
Texas A&M University
Rock-eating microbes survive in deep ocean off Peru
Rock-eating microbes survive in deep ocean off Peru Way
down deep in the ocean off the coast of Peru, in the rocks
that form the sea floor, live bacteria that don't need sunlight,
don't need carbon dioxide, don't need oxygen. These
microbes subsist by eating the very rocks they call home.
Researchers from the Ocean Drilling Program (ODP) have
embarked aboard the world's largest scientific drillship on a
voyage to understand the abundance and diversity of these
microbes and the environments in which they live.
Biogeochemical Redox Couples
What is the energy currency of metabolic
reactions in cells ??
How do cells make it ?
What powers those reactions?
How do we measure the energy outputs or
requirements of metabolism?
How can we use this kind of information in an
ecological and biogeochemical sense?
Biogeochemical Redox Couples

CO2 + H2O  CH2 O + O2
oxygenic photosynthesis
Interdependency?
CH2 O + O2  CO2 + H2O (+)
CH4 + 2O2  CO2 + 2H2O(+)
CO2 +
HS-+

aerobic respiration
oxidative methanotrophy
H2O  biomass + SO42-
C6H12 O6  2CO2 + 2C2H6O (+ )
anoxygenic
photosynthesis
fermentation
4H2+SO42-  S2-+ 4H2O (+)
sulfate reduction
CO2 +2H2  CH4 + 2H2O (+)
methanogenesis
Redox Potentials
& Energy Yields
The electron tower……..
Strongest reductants, or e donors,
on top LHS
Electrons ‘fall’ until they are
‘caught’ by available acceptors
The further they fall before being
caught, the greater the difference
in reduction potential and energy
released by the coupled reactions
(Last Common Ances
Redox Potentials
& Energy Yields
The energetically most favored
The energetically most favored
reaction proceeds first ie
CH2O first degraded with O2
CH2O degraded with NO3 nex
CH2O degraded with Mn4+ next
followed by SO42-,
and CO2 last (methanogenesis)
Energy Calculations
aA +bB ‡ cC + cD
G = Gf°’ (aA + bB) – Gf°’ (cC + dD)
Where Gfo’ is the free energy of formation of 1 mole
under ‘standard’ conditions (pH 7, 25C)
G = G° (T) + RT·ln K
.
K=CcDd/AaBb R= 1.98cal.mol-1.°K-1
G = G° (T) +RT·ln
[C]c[D]d
[A]a[B]b
Organic compound
How do microbes garner energy and
carbon?
Carbon flow
Electron flow
respiration
Organic compound
Carbon flow
Electron flow
anaerobic respiration
Other organic compound
Inorganic compound
H2 H2S NH2 Fe2+
Carbon flow
Electron flow
lithotrophy
Biosynthesis
Mechanisms and
Balance Sheets
Electron Donor
Electron “Carrier”
NAD + H2 ‡ NADH
(catab)
or
Terminal Electron Acceptor
NADP + H2  NADPH
(anab)
Balance Sheet: pyruvic acid  3CO2 = 4 NADH + 1 FADH (Flavoproetein e carrier)
1NADH  3 ATP; 1FADH  2ATP therefore 1 TCA cycle  15ATP; 1 glucose  30ATP
1ATP  7kcal/mole so 1 molecule glucose  266 kcal
Glucose oxidation with O2 G = 688kcal Therefore aerobic respiration ca. 39% efficient
In contrast, glucose fermentation  lactate = 29 kcal/mol ca. 50% efficient
Reactions of the TCA Cycle
Pyruvate
The TCA cycle showing enzymes, substrates and products. The abbreviated enzymes are:
IDH = isocitrate dehydrogenase and a-KGDH = a-ketoglutarate dehydrogenase. The
GTP generated during the succinate thiokinase (succinyl-CoA synthetase) reaction is
equivalent to a mole of ATP by virtue of the presence of nucleoside diphosphokinase.
The 3 moles of NADH and 1 mole of FADH2 generated during each round of the cycle feed
into the oxidative phosphorylation pathway. Each mole of NADH leads to 3 moles of ATP
and each mole of FADH2 leads to 2 moles of ATP. Therefore, for each mole of pyruvate
which enters the TCA cycle, 12 moles of ATP can be generated
Balance Sheet:
pyruvic acid  3CO2 = 4 NADH + 1 FADH (Flavoproetein e carrier)
1NADH  3 ATP; 1FADH  2ATP therefore 1 TCA cycle 15ATP; 1
glucose  30ATP
1ATP  7kcal/mole so 1 molecule glucose  266 kcal
Glucose oxidation with O2 G = 688kcal Therefore, in this case, aerobic
respiration ca. 39% efficient
In contrast, glucose fermentation  lactate = 29 kcal/mol ca. 50%
efficient
Multi-Step Organic Matter Remineralization in Anoxic Systems
Biopolymers
(CH2O)n
NO3-  NH4+
Mn4+  Mn2+
Monomers
Fe3+  Fe2+
Small Organics
So42-  H2S
CO2
CO2  CH4
oxidation
reduction
Requires numerous extracellular electron transfers
H2 
+
2H +
2e
A nearly ubiquitous means of
extracellular electron transport in
microbial redox chemistry
Hydrogen
• Anaerobic metabolism strongly sensitive to pH2
• Fermentation frequently characterized by obligate
(1-2 C’s) or facultative (>3 C’s) H2 production •
•Reaction only energetically feasibly with H2 sink
•Obligate H2 producers don’t grow in ‘pure’ culture
•Readily grown in co-culture
•H2 consuming reactions affected oppositely
Hydrogen
• H2 consuming reactions affected oppositely
e.g. with mM SO42-SRB can maintain very
low pH2.
• In presence of active SRB, H2 too low for methane
production to be energetically feasible
• Often see zonation between SR and MP under
thermodynamic control
Hydrogen
• 2H2 + 2CO2  CH3COOH + O2 + G
• CH3COOH + O2  2H2 + 2CO2 + G
Opposite biochemistry when methanogen present
Anaerobic oxidation of methane is energetically marginal
unless????
• 2CH4 +SO42-  S2-+ 2CO2 +4H2
H2 has a High Relative Stoichiometry in Many
Anaerobic Remineralization Processes
Production
CH3CH2COOH + 2H2O  CH3COOH + CO2 + 3H2
Consumption
CO2 +4H2  CH4 +2H2O
Free Energy Yield Depends Exponentially on
Stoichiometry in Reaction
CO2 +4H2  CH4 +2H2O
G= G°(T)+RT‧In
PCH4
PCO2(PH2)4
Gmp is much more sensitive to PH2 than to PCH4 or PCO2
Thermodynamics of Inter-Species H2 Transfer
producer
Both Organisms Depend Highly
on H2 Partial Pressure:
Too High Alters Production
 Pathway Shifts, Inhibition, Reversa
consumer
Too Low Inhibits
Consumption
H2 in the Environment
producer
PH2 controlled by the balance
between production and
consumption
For constant or decreasing H2
production rate (e.g. sediments), PH2
in practice reflects control by H2
consumption
consumer
Consumption very efficiently
coupled to production; PH2 held at
very low steady-state levels;
residence times short (seconds or
less)
Free Energy Regulation in Methanogenesis
4H2 + CO2 
CH3COOH 
CH4 + 2H2O
CH4 + CO2
Data for methanogenic sediments from Cape Lookout Bight at 22°C;
Responsiveness [X] and Dt required to change free energy yield by 10kJ/mole
Inter-Species H2 Transfer
in a Complex Microbial Ecosystem
producer2
producer1
comsumer 1
H2
comsumer 3
producer3
comsumer 2
Controls on Hin Anoxic Sediments
producer
consumer
PH2 in sediments is controlled
by H2 consumers
Steady-state PH2 reflects efficiency
of consumption; constrained by
physiologic limitations of H2
consumers
Ultimate physiologic limitation:
requirement to extract sufficient free
energy from H2 consumption to permit
continued metabolism
Steady State H2 Concentrations Sensitive To:
Concentrations of Products and Reactants (Xox and Xred)
Specific Redox Couple (e.g. CO2/CH4 -vs- SO42-/S2-)
Temperature
Energy Yield of Reaction (Grxn)
Effect of Sulfate Concentration on H2
SO42- + 4H2  S2- + 4H2O
Increasing Sulfate = Decreasing H2
Impact of Sulfate Concentration Change on DG and H2
in Sulfate-Reducing CLB Sediments
G
H2
Expected GSR-vs-SO42-
Sulfate (mM)
Sulfate (mM)
Deduction: H2 is drawn down to compensate for increasing sulfate; SRB community
Maintainconst G near limit for ‘maintenance’ but max efficiency. An adaptation to
substrate limitation?
Depth Profiles of H2 in CLB Sediments
Sulfate (mM)
Sulfate (mM)
Sulfate
Sulfate
Depth
(cm)
August
27oC
H2 (Pa)
November
14.5oC
H2 (Pa)
Inter-Species H2 Transfer
in a Complex Microbial
comsumer 1
affecting G
etc.)
Both can be
Address
Quantitatively
comsumer 2
Bulk phase (extracellular) H2 partial pressures are described
quantitatively by intracellular thermodynamics
P
2
H
Extracellular
Measurement
Intracellular
Bioenergetics
Spatial Constraints
H2
H2
consumer
(HC)
H2
consumer
Organic
matter
producer
producer
(HP)
H2 measurement
HP
bulk fluid HC
PH2 measured in bulk fluid > PH2 in HC cell
(HC)
(HP)
H2 measurement
HP HC bulk fluid
PH2 measured in bulk fluid = PH2 in HC cell
Efficient utilization of H2 requires mass transport and high concentration gradient unless
mitigated by spatial arrangements. The fact that quantitative H2 etc measurements reflect
bioenergetic control argues for non-random arrangement of consumers and producers as
illustrated above (see later re AOM)
In Situ Free Energy Yields in CLB Sediments
G(KJ·mol-1)
MP
MP
Depth (cm)
G(KJ·mol-1)
August
T=27oC
November
T=14.5oC
Biogeochemical Redox Couples
aerobic respiration
CH2 O + O2  CO2 + H2O
1 mole glucose
30-32 mole ATP
1 mole glucose
2-4 mole ATP
Biosynthesis requires approx. 1mole ATP per 4g of cell carbon
Biogeochemical Redox Couples
oxygenic photosynthesis
CO2 + H2O  CH2 O + O2
Molecule of the Month
http://www.bris.ac.uk/Depts/Chemistry/MOTM/atp/atp1.htm
Adenosine Triphosphate - ATP
Paul May – Bristol University
The 1997 Nobel prize for Chemistry has been awarded to 3 biochemists for the study
of the important biological molecule, adenosine triphosphate . This makes it a fitting
molecule with which to begin the 1998 collection of Molecule's of the Month. Other
versions of this page are: a Chime version and a Chemsymphony version.
ATP - Nature's Energy Store
All living things, plants and animals, require a continual supply of energy in order to function.
The energy is used for all the processes which keep the organism alive. Some of these
processes occur continually, such as the metabolism of foods, the synthesis of large,
biologically important molecules, e.g. proteins and DNA, and the transport of molecules and
ions throughout the organism. Other processes occur only at certain times, such as muscle
contraction and other cellular movements. Animals obtain their energy by oxidation of foods,
plants do so by trapping the sunlight using chlorophyll. However, before the energy can be
used, it is first transformed into a form which the organism can handle easily. This special
carrier of energy is the molecule adenosine triphosphate, or ATP
Its Structure
ribose (the
same sugar that forms the basis of DNA). Attached to one side of this is a base (a
group consisting of linked rings of carbon and nitrogen atoms); in this case the
base is adenine. The other side of the sugar is attached to a string of phosphate
groups. These phosphates are the key to the activity of ATP.
The ATP molecule is composed of three components. At the centre is a sugar molecule,
ATP consists of a base, in
this case adenine (red), a
ribose (magenta) and a
phosphate chain (blue).
AMP
ADP
ATP
How it works
ATP works by losing the endmost phosphate group when instructed to do so by
an enzyme. This reaction releases a lot of energy, which the organism can then
use to build proteins, contact muscles, etc. The reaction product is adenosine
diphosphate (ADP), and the phosphate group either ends up as orthophosphate
(HPO4) or attached to another molecule (e.g. an alcohol). Even more energy can
be extracted by removing a second phosphate group to produce adenosine
monophosphate (AMP)
ATP + H2O  ADP + HPO4
When the organism is resting and energy is not immediately needed, the reverse
reaction takes place and the phosphate group is reattached to the molecule
using energy obtained from food or sunlight. Thus the ATP molecule acts as a
chemical 'battery', storing energy when it is not needed, but able to release it
instantly when the organism requires i
The 1997 Nobel Prize for Chemistry
The Nobel prize for Chemistry in 1997 has been shared by:
Dr John Walker of the Medical Research Council's Laboratory of Molecular Biology (LMB)
at Cambridge (an institution which has been responsible for 10 Nobel prizes since 1958!)
Dr Paul Boyer of the University of California at Los Angeles
and Dr Jens Skou of Aarhus University in Denmark.
The prize was for the determination of the detailed mechanism by which ATP shuttles
energy. The enzyme which makes ATP is called ATP synthase, or ATPase, and sits on the
mitochondria in animal cells or chloroplasts in plant cells. Walker first determined the amino
acid sequence of this enzyme, and then elaborated its 3 dimensional structure. Boyer
showed that contrary to the previously accepted belief, the energy requiring step in making
ATP is not the synthesis from ADP and phosphate, but the initial binding of the ADP and the
phosphate to the enzyme. Skou was the first to show that this enzyme promoted ion
transport through membranes, giving an explanation for nerve cell ion transport as
well as fundamental properties of all living cells. He later showed that the phosphate group that is
ripped from ATP binds to the enzyme directly. This enzyme is capable of transporting
sodium ions when phosphorylated like this, but potassium ions when it is not. More details
on the chemistry of ATPase can be found here, and you can download the 2 Mbyte pdb file
for Bovine ATPase from here.
References: Chemistry in Britain, November 1997, and much more information on the
history of ATP and ATPase can be found at the Swedish Academy of Sciences and at
Oxford University.