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Histidine-Lysine Axial Ligand Switching in a Hemoglobin: A Role for Heme Propionates.

ABSTRACT: The hemoglobin of Synechococcus sp. PCC 7002, GlbN, is a monomeric group I truncated protein (TrHb1) that coordinates the heme iron with two histidine ligands at neutral pH. One of these is the distal histidine (His46), a residue that can be displaced by dioxygen and other small molecules. Here, we show with mutagenesis, electronic absorption spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy that at high pH and exclusively in the ferrous state, Lys42 competes with His46 for the iron coordination site. When b heme is originally present, the population of the lysine-bound species remains too small for detailed characterization; however, the population can be increased significantly by using dimethyl-esterified heme. Electronic absorption and NMR spectroscopies showed that the reversible ligand switching process occurs with an apparent pKa of 9.3 and a Lys-ligated population of ∼60% at the basic pH limit in the modified holoprotein. The switching rate, which is slow on the chemical shift time scale, was estimated to be 20-30 s-1 by NMR exchange spectroscopy. Lys42-His46 competition and attendant conformational rearrangement appeared to be related to weakened bis-histidine ligation and enhanced backbone dynamics in the ferrous protein. The pH- and redox-dependent ligand exchange process observed in GlbN illustrates the structural plasticity allowed by the TrHb1 fold and demonstrates the importance of electrostatic interactions at the heme periphery for achieving axial ligand selection. An analogy is drawn to the alkaline transition of cytochrome c, in which Lys-Met competition is detected at alkaline pH, but, in contrast to GlbN, in the ferric state only.

SUBMITTER: Nye DB

PROVIDER: S-EPMC6214620 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 ?Fe-His and ?CO 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 an 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.

Dataset Information

Histidine-Lysine Axial Ligand Switching in a Hemoglobin: A Role for Heme Propionates.

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