Chemistry Reference
In-Depth Information
A current working model for ISC biogenesis in eukaryotes is illustrated in
Fig. 4.11
.
The names used are those
of the human components. The biogenesis involves the transient de novo synthesis of an ISC, with the sulfur atom
derived from cysteine via the pyridoxal phosphate (PLP)-dependent cysteine desulfurase, Nfs1 in complex with
Isd11. In this reaction, the sulfur atom of free cysteine is transferred to a conserved cysteine of Nfs1 to form
a persulfide as a reaction intermediate. The sulfur is then transferred directly to the scaffold protein complex
ISCU, a necessary process to avoid the potential unregulated release of toxic sulfide. The reduction of sulfur to
sulfide requires electrons derived from NADH (Nicotinamide Adenine Dinucleotide, in its reduced form) via an
electron-transfer chain involving the ferredoxin reductase (FdxR) and the [2Fe
2S] ferredoxin (Fdx). Iron enters the
mitochondria through the carrier protein mitoferrin (Mfrn1/2) and, in a process involving the iron chaperone protein,
frataxin (Fxn), is combined with sulfide on the mitochondrial Fe/S scaffold ISCU. The Fe/S cluster transiently bound
to ISCU is then transferred to mitochondrial apoproteins in an ATP-dependent process. This requires a chaperone
system (consisting of the Hsp70 chaperone Grp75, HscB, and GrpE) and the monothiol glutaredoxin GLRX5.
Additional proteins, Isa1, Isa2, and Iba57, play specific roles in delivering Fe/S clusters to a subset of Fe/S proteins that
includes aconitase-like and radical S-adenosyl methionine (SAM) Fe/S proteins, whereas Ind1 is involved in the
assembly of Fe/S clusters in respiratory complex I (see Chapter 5). For cytosolic and nuclear Fe/S proteins, an
unknown intermediate (X) is synthesised by the ISC assembly machinery and transported out of the mitochondria by
the inner membrane ABC transporter
8
ABCB7. The process seems to require both the sulfhydryl oxidase ALR and
glutathione (GSH). In the cytosol, a transiently bound Fe/S cluster is formed on a scaffold complex consisting of the P-
loop NTPases Cfd1 and Nbp35. The Fe/S clusters are then transferred and inserted into extramitochondrial apo-
proteins in a reaction involving IOP1 and CIAO1.
e
MORE COMPLEX COFACTORS
e
MOCO, FEMOCO, P-CLUSTERS, H-CLUSTERS, AND CUZ
Our understanding of metal incorporation into metalloporphyrins and Fe
e
S clusters, which are widely
distributed in a great many metalloproteins has advanced greatly in recent years. However, it has also become
apparent that there are a growing number of more complex cofactors, some of them with a more specific
distribution. The transition metal molybdenum (Mo) is found as an essential part of the active site in a wide
range of metalloenzymes in bacteria, fungi, algae, plants, and animals. However, the metal itself is biologically
inactive unless it is incorporated into a special molybdenum cofactor (MoCo), which incorporates a dithiolene
group (
Fig. 4.6
a
) and is required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine
dehydrogenase, and aldehyde oxidase. In all organisms studied to date, MoCo is synthesised by a highly
conserved biosynthetic pathway (
Fig. 4.12
)
, which can be divided into five steps involving the biosynthetic
intermediates precursor cyclic pyranopterin monophosphate (cPMP), MPT (Metal Binding Pterin), adenylated
MPT, and MoCo. The six enzyme activities involved in MoCo biosynthesis (and their corresponding genes) have
been widely identified. As is common in the biosynthesis of other flavins and pterins, MoCo synthesis starts
from guanosine triphosphate (GTP), and involves the circularisation of GTP to the precursor cyclic pyranopterin
monophosphate (cPMP). This reaction is catalysed by two proteins, one a radical SAM
9
enzyme and the second
protein involved in pyrophosphate release. The former has two oxygen-sensitive [4Fe
4S] clusters, one of
which is involved in radical SAM generation while the other is crucial for substrate binding. Thereafter, three
enzymes are responsible for formation of the dithiolene group in the metal-binding pterin molybdopterin (MTP).
Two sulfur atoms are incorporated into cPMP by the heterotetrameric (two large subunits and two small
subunits) enzyme MPT synthase to yield MTP, with its characteristic dithiolate function. The small subunit
carries a sulfur atom as a thiocarbonate at its C-terminal Gly residue, which is deeply buried in the large subunit
to form the active site. Unusually, in human MTP synthase both subunits are encoded by a biscistronic mRNA.
e
8. Membrane transport proteins having the ABC molecular domain (ATP-binding cassette), characteristic of a large superfamily of proteins
that hydrolyse ATP and transport a diverse array of small molecules across membranes.
9. Radical SAM (S-adenosyl methionine) enzymes utilise iron
e
sulfur clusters and SAM to initiate a diverse set of radical-mediated reactions.
