Investigations of Heme Ligation and Ligand Switching in Cytochromes P450 and P420

Abstract

It is generally accepted that the inactive P420 form of cytochrome P450 (CYP) involves the protonation of the native cysteine thiolate to form a neutral thiol heme ligand. On the other hand, it has also been suggested that recruitment of a histidine to replace the native cysteine thiolate ligand might underlie the P450→P420 transition. Here we discuss resonance Raman investigations of the H93G myoglobin (Mb) mutant in the presence of tetrahydrothiophene (THT) or cyclopentathiol (CPSH), and on pressure-induced cytochrome P420cam (CYP101), that show a histidine becomes the heme ligand upon CO binding. The Raman mode near 220 cm−1, normally associated with the Fe-histidine vibration in heme proteins, is not observed in either reduced P420cam or the reduced H93G Mb samples, indicating that histidine is not the ligand in the reduced state. The absence of a mode near 220 cm−1 is also inconsistent with a generalization of the suggestion that the 221 cm−1 Raman mode, observed in the P420-CO photoproduct of inducible nitric oxide synthase (iNOS), arises from a thiol-bound ferrous heme. This leads us to assign the 218 cm−1 mode observed in the 10 ns P420cam-CO photoproduct Raman spectrum to a Fe-histidine vibration, in analogy to many other histidine bound heme systems. Additionally, the inverse correlation plots of the νFe-His and νCO frequencies for the CO adducts of P420cam and the H93G analogs provide supporting evidence that histidine is the heme ligand in the P420-CO bound state. We conclude that, when CO binds to the ferrous P420 state, a histidine ligand is recruited as the heme ligand. The common existence of a HXC-Fe motif in many CYP systems allows the C→H ligand switch to occur with only minor conformational changes. One suggested conformation of P420-CO involves the addition of another turn in the proximal L helix so that, when the protonated Cys ligand is dissociated from the heme, it can become part of the helix and the heme is ligated by the His residue from the adjoining loop region. In other systems, such as iNOS and CYP3A4 (where the HXC-Fe motif is not found) a somewhat larger conformational change would be necessary to recuit a nearby histidine.

Redox-dependent Ligand Switching in a Sensory Heme-binding GAF Domain of the Cyanobacterium Nostoc sp. PCC7120

“…The positions of other prominent core marker bands (e.g. 2 and 10 ) are also consistent with a 6cLS heme (39). The CϭC modes characteristic of the heme vinyl substituents are well separated (1615 and 1627 cm Ϫ1 ), indicating a rather different environment of the vinyl groups of rings A and B (36,40).…”

“…Hemes, 6 in particular protoheme, are ubiquitous and essential protein cofactors for many biological reaction pathways (1)(2)(3)(4). Nature has evolved quite variable protein folds competent for heme incorporation, in a covalent or noncovalent manner.…”

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…When the thiolate ligand is protonated to form a neutral thiolate-heme ligand or in case the thiolate ligand is replaced by a histidine, resulting a nitrogen-bound form, a peak at 420 nm is observed [48].…”

Rapid kinetic methods to dissect steroidogenic cytochrome P450 reaction mechanisms

“…cysteine-ligated P450s catalyze RH → ROH, and histidine-ligated peroxidases catalyze ROOR → 2ROH). Additionally, the axial cysteine residue in P450 enzymes is the reason for the 450 nm Soret band upon CO binding [32]. If the axial cysteine residue is mutated to a histidine, then the Soret band appears at 420 nm [33].…”