Cytochrome c

Cytoplasmic chromophore100 amino acids, 12 kDa. Heme Fe coordinated by His and Met.

Single electron transfer.

Covalently attached to the protein at Cys side chains, via heme propionates.

E°’ = 250 mV

http://en.wikipedia.org/wiki/Cytochrome_c A.-F. Miller, 2008, pg 1 Heme Fe

A.-F. Miller, 2008, pg 2 Garrett & Grisham, Figure 5.31 O2 vibrational frequencies (how to know its effective oxidation state) -1 Species νO-O(cm ) dO-O(Å)

+ O2 1,905 1.12

O2+ 1,580 1.21

- O2 1,097 1.33

2- O2 802 1.49

A.-F. Miller, 2008, pg 3 II O2 binding to Fe is associated with electron transfer From resonance Raman spectroscopy, the O-O stretch in -1 5 III - oxyMb is ≈ 1,105 cm , protein is diamagnetic, d , Fe -O2

A.-F. Miller, 2008, pg 4 A.-F. Miller, 2008, pg 5 Garrett & Grisham, Fig. 20.10, 20.09 Myoglobin

A.-F. Miller, 2008, pg 6 Cytochrome P450

Cytoplasmic chromophore absorbing at 450 nm Also: Chloroperoxidase and NO synthase, peroxidase.

- + RH + O2 + 2 e + 2 H → ROH + H2O Heme Fe has one protein ligand, from Cys S-.

E°’m = -220, changes to -140 upon substrate binding. Metabolism of xenobiotics, activation of drugs, hormones and prostaglandins.

Chloroperoxidases (such as myeloperoxidase) use H2O2 as the O source: immune function. A.-F. Miller, 2008, pg 7 Substrate binding to the protein is associated with Mechanism release of water from second Fe3+ coordination site and H H conversion of the heme from O low-spin to high-spin.

Fe3+ can then be reduced more easily to Fe2+ and bind O2. O

Peroxo intermediate converts to FeIV=O 2 intermediate. ⇔ III - A.-F. Miller, 2008, pg 8 Fe -OO ‘Peroxide shunt’ Mechanism The fact that addition of H H O peroxide achieves the same intermediate as is formed upon O2 addition to reduced enzyme shows that the O enzyme gives those two reducing 2 equivalents to O2. ⇔ III - A.-F. Miller, 2008, pg 9 Fe -OO ‘Compound one’ and ‘compound two’

Cpd I formed from H2O2 + P450 When a remote amino acid is the source of the other electron (and becomes a radical: Cytochrome c peroxidase uses a Trp•), then the spectral properties are those of CpdII. Cpd II is reduced by one electron relative to Cpd I

A.-F. Miller, 2008, pg 10 Spin state change and oxidation state change are coupled to substrate binding

Das et al. (2007) J.A.C.S. 129:13378-13379

A.-F. Miller, 2008, pg 11 Das et al. (2007) JACS 129:13378-13379

A.-F. Miller, 2008, pg 12