Oxidative Phosphorylation II

2017-11-20

Conceptual goals

• Understand how the electron transport chain couples movement of electrons along a REDOX gradient to creating a proton gradient
• Understand how different cofactors can safely shuttle electrons

Skill goals

• Reason about the linkage between changes in reduction potential and ability to transport protons against a concentration gradient
• Reason about the relative ordering of the electron transport chain based on REDOX potentials and perturbation with drugs

Oxidative phosphorylation overview:

Why does oxidative phosphorylation break the $NADH + \frac{1}{2}O_{2} + H^{+} \rightarrow NAD^{+} + H_{2}O$ reaction into multiple steps?

Control, capture of energy at each step

Where does ox-phos occur (and which way are protons pumped)?

In the mitochondria, pumping protons from matrix into intermembrane space

Complex I couples oxdiation of $Q$ to $QH_{2}$ to transport of protons across the bilayer

Process has many sequential steps

$NAD:H + H^{+} + FMN \rightarrow FMNH_{2}$

$FMNH_{2} + 2Fe^{3+} \rightarrow FMN + H_{2} + 2Fe^{2+}$

Pass along several iron-sulfur clusters

$2Fe^{3+} + 2Fe^{2+} \rightarrow 2Fe^{2+} + 2Fe^{3+}$

$2Fe^{2+} + Q + 2H^{+} \rightarrow 2Fe^{3+} QH_{2}$

FMN accepts electrons directly from NADH

Iron-sulfur clusters

Iron-sulfur clusters

• Driven by oxidation of $Fe^{2+}$ to $Fe^{3+}$.
• Single electron transfer
• Sulfur complexes hold $Fe$ iron away from water and physically links oxidation state of $Fe$ to rest of protein

The mysterious $Q$ (ubiquinone, coenzyme Q10)

Ubiquinone

• Driven by oxidation carbonyl/hydroxyls on conjugated ring

• Two electron transfer
• Both oxidized and reduced forms are uncharged. Diffuses in the bilayer

Key point: the $2H^{+}$ come from the matrix side

Transfer is also linked to transfer of four more protons...somehow.

Complex I is not the only source of $QH_{2}$.

Succinate enters oxidative phosphorylation via Complex II, generating $QH_{2}$

Fatty acid oxidation, other oxidation reactions can generate $QH_{2}$ using a variety of enzymes

What happens to $QH_{2}$?

Enters "Q-cycle" which ultimately oxidizes cytochrome C and moves $4H^{+}$ across the bilayer

$2QH_{2} \rightarrow QH_{2} + Q + 2H^{+}$

Key point: this is an indirect pump. Geometry of complex III means protons are preferentially picked up on matrix side, preferentially dropped on lumen side

Oxidative phosphorylation overview:

Cytochromes

• Driven by oxidation of $Fe^{2+}$ to $Fe^{3+}$.
• Single electron transfer
• Heme holds $Fe$ iron away from water and physically links oxidation state of $Fe$ to rest of protein

Cytochromes hold onto this heme ring in a deep pocket, protecting Fe

Cytochrome c is a "peripheral" membrane protein that "skates" over to complex IV

Oxidative phosphorylation overview:

Complex IV uses $O_{2}$ to oxidize $CytC(Fe^{2+})$.

$4\ CytC(Fe^{2+}) + O_{2} + 4H^{+} \rightarrow 4\ CytC(Fe^{3+}) + 2H_{2}O$

This transports 2 more protons across the bilayer

Why might succinate to fumarate come in to ox-phos directly as $QH_{2}$ (rather than going through complex I)?

Does this give as much energy as an NADH?

Highest yield step?

$QH_{2} \rightarrow cytochrome\ C$

Surprising?

Why not transport more $H^{+}$ at the $O_{2}$ step? I'm not sure, actually

Why might electron transfers be so tightly controlled?

Premature contact with $O_{2}$ can generate highly reactive $\cdot O_{2}^{-}$ radical.

Summary I

• Passage through Complexes I-IV allows oxidation of $NADH$ and other biomolecules, ultimately reducing $O_{2}$.
• This transports a total of 10 protons out of mitochondrial matrix
• This "recycles" $NADH$ and $QH_{2}$ into their oxidized forms

Summary II

• REDOX centers (Fe-S, hemes, Q, cytochromes) allow ordered, vectorial movement of electrons towards increased reduction potentials
• Integral redox centers couple electron transport to allosteric conformational changes in complexes
• Allosteric conformational changes in complexes lead to transport of protons