Hang onto your haem…what's happened to CYP4A?

Abstract

It is getting even more difficult to generalize about the cytochrome P450 enzymes – a diverse superfamily of enzymes that are ubiquitous in nature and have key roles in xenobiotic metabolism and steroid biosynthesis in mammals. It used to be easy to show a common theme in their basic architecture at least, and to describe how they differed structurally from almost all other haemoproteins. P450s have a characteristic way of attaching the prosthetic haem group to the protein, via coordination to a cysteine residue that is highly conserved in all members of this huge superfamily of proteins. This is what gives P450s their characteristic absorption spectrum, with the peak at ∼450 nm in the reduced and carbon-monoxide-bound state. Now, it seems that there is an additional means of tethering the haem in at least one group of P450s, the CYP4A family, which might liken these enzymes to certain peroxidases, another group of haemoproteins that shares some functional similarities to P450s.The CYP4A family of enzymes are responsible for the metabolism of fatty acids at the terminal position of the hydrocarbon chain. Hoch and Ortiz de Montellano 1xSee all References1 found that during certain analytical techniques in which the haem usually separates from the protein, the prosthetic group appeared to remain attached, at least partially, to the apoprotein. The degree of haem association varied from 6% to 97% among several recombinant CYP4A forms. Intriguingly, CYP4A1 purified from rat liver appeared to show about 80% haem binding (which was more than the cognate recombinant form), suggesting that the covalent attachment occurs naturally and not just in the artificial expression system. Mass spectrometric analysis suggested that the haem was attached through an ester linkage between an unusual hydroxyl group on the haem and a glutamic acid residue in the protein. There is a precedent for this: in certain mammalian peroxidases the haem group is doubly hydroxylated and linked at two points via ester linkages to acidic residues of the protein. The linkage appears to occur in the first enzymatic turnover.The outstanding question is: does covalent attachment of the haem affect the activity of the CYP4A enzymes? There are hints that it might; mutants in which attachment was lost (and the haem was not hydroxylated) exhibited considerably reduced (or undetectable) catalytic activity and substrate binding. At this stage it is impossible to tell whether the extra attachment is necessary or beneficial for activity, or just an accidental side-effect of having an active enzyme. Why it might be useful to have a second site of attachment for the haem in CYP4A is anyone's guess, but the possibility that this serves a functional role in CYP4As reveals just how little we understand about events at the catalytic heart of these intriguing enzymes.