Microreview Hydrogenases: FeNi, Fe-only and non-Fe. All contain Fe.

Fe-only has a pair of Fe coupled to an Fe4S4 cluster by a Cys S. This is the only ligand to the Fe2S2 sub-cluster derived from the . Fes are Oh with S2- bridges, + - CN/CO and a ‘’. H2 ⇔ 2 H + 2 e chemistry is proposed to occur on terminal Fe. non-Fe hydrogenase employs a methylene + - tetrahydrobiopterin as the site of H2 ⇔ 2 H + 2 e chemistry. An Fe nonetheless stands nearby, again a low-spin Fe with CN and CO ligands. ??Side-on coordination of H2 or Lewis acid action to favour heterolysis of H2 ?

A.-F. Miller, 2008, pg 1 e- injected by the Fe-protein which couples et to ATP . e- go to the P cluster in the MoFe-protein, which then transfers them to the catalytic site on the FeMoco. The identity of a central atom inside the FeMoco remains a mystery (watch the literature !!). binds and is converted stepwise to product via binding to an Fe on one face of the FeMoco.

A.-F. Miller, 2008, pg 2 Comparison with other hydrogenases

Shima et al. 2008 Science 321: 572 A.-F. Miller, 2008, pg 3 The FeS clusters involved

A.-F. Miller, 2008, pg 4 Howard & Rees, (2006) PNAS 103:17088 A.-F.Barney Miller, 2008, et alpg (2006)5 PNAS 103:17113 Alternative mechanisms, inspired by Chatt.

Howard & Rees, (2006) A.-F. Miller, 2008, pg 6 PNAS 103:17088 Formation of Fe/S centres

Fe/S cluster formation represents very favourable chemistry (R. Holm) but this would need to be protected from O2 and H2O. Similar considerations apply to clusters containing Mo, V and Ni. Nonetheless, Fe/S clusters are found in virtually all biological systems. Origin in circumstances where all systems were in equilibrium + - 2- - - with H2 ⇔ 2H + 2e and S ⇔S↓ + 2e , where e would come and go from .

A.-F. Miller, 2008, pg 7 Ancient origins again

The large e-t complexes of mitochondria and chloroplasts that contain Fe/S clusters also include at least one polypeptide each that is encoded by the organelle genome. One does not see excess Fe, S or peptides: implication that uptake and protein expression are co-ordinated. !!?? the coordination site of Zn in Zn fingers looks favourable for Fe2+ as well. Significance of cavity size and second sphere residues.

A.-F. Miller, 2008, pg 8 IscU, [Fe2S2]-IscU, [Fe2S2]IscU•IscS, 67 uM each.

Dennis Dean, (2008) PNAS 105(25) 8591-8596.

A.-F. Miller, 2008, pg 9 Cluster assembly

Cys-S~S

IscS IscU

IscU is the assembly scaffold. S-trafficking PLP-dep Cys desulfurase IscS, gives S to IscU

A.-F. Miller, 2008, pg 10 Non-heme, Non-S Fe centres

O Fe Fe Fe O

Execute a huge range of chemistry, ‘possibly’ exceeding that of hemes. Found in diverse protein scaffolds (although some are particularly common). Redox catalysts, with a particularly important roles in activating O2.

A.-F. Miller, 2008, pg 11 Why does O2 need activating ?

Conservation of spin: total spin of products = total spin of reagents.

For glucose + O2 → 6CO2 + 6H2O S = 0 + 1 → 0 + 0 (Radical chain reactions produce a new radical for each parent radical that reacts: reagents Stot = 1/2, product Stot = 1/2). A cofactor with access to multiple spin states in needed to effect spin conversion from (for example) S=2 + S=1 → S=5/2 + S=1/2 → S=5/2 - S=1/2 → S=2 + S=0.

A.-F. Miller, 2008, pg 12 Mononuclear Fe2+ in a HXD/ E . . .H facial triad.

In DSBH, first two lig at end of second strand, third lig from 7th strand. There are variations though. A.-F. Miller, 2008, pg 13 Koehntop and Que, Jr. (2005) JBIC 10:87-93. Koehntop and Que, Jr. (2005) JBIC 10:87-93.

A.-F. Miller, 2008, pg 14 Active sites of mononuclear NH Fe Common ligation, diferent substrate-binding modes Naphtahlene 1,2- 2,3-dihydroxybiphenyl dioxygenase 1,2 dioxygenase (Rieske)

IPNS

Clavaminate synthase phenylalanine hydroxylase (pterin)

A.-F. Miller, 2008, pg 15 Koehntop and Que, Jr. (2005) JBIC 10:87-93. O2 binding ?

The last thing that binds is O2. Binding is side-on for NDO (the only structure of an authentic O2-adduct).

Bph dioxygenase Isopenicillin-N-synthase Clavaminate synthase

A.-F. Miller, 2008, pg 16 2-oxoacid dependent dioxygenases

A large variety of reactions

Many employ not only O2, but also a 2-oxo acid, such as 2- oxoglutarate (αketoglutarate).

A.-F. Miller, 2008, pg 17 Clifton et al (2006) J. Inorg. Biochem 100:644-669. 2OA-dependent oxygenases Oxygenases insert an O atom from O2 into product. (oxidases need not). Di-oxygenases insert both O2 atoms.

In 2-OG-dependent oxygenases, one O from O2 reacts with 2OA, other inserts into product.

O O O O- - O O- O - O O O O O O O

-O O -O O O -O Fe2+ O Fe3+ Fe4+

O O

O- Very reactive !! O- -O O O O C O O O Fe4+ O A.-F. Miller, 2008, pg 18 Fe4+ A.-F. Miller, 2008, pg 19 Clifton et al (2006) J. Inorg. Biochem 100:644-669. Binding of substrate usually precedes binding of O2 and converts 6-coordinate Fe2+ to five-coordinate. 2+ O2 can then bind and be reduced and activated by Fe .

A high-spin FeIV intermediate has been trapped for TauD. The chemistry taht follows is very diverse and highly favourable energetically.

In the absence of substrate, the protein can be modified, and fragmented.

A.-F. Miller, 2008, pg 20