The H93G myoglobin cavity mutant as a versatile scaffold for modeling heme iron coordination structures in protein active sites and their characterization with magnetic circular dichroism spectroscopy

Abstract

Preparation of heme model complexes is a challenging subject of long-standing interest for inorganic chemists. His93Gly sperm whale myoglobin (H93G Mb) has the proximal His replaced with the much smaller non-coordinating Gly. This leaves a cavity on the proximal side of the heme into which a wide variety of exogenous ligands can be delivered. The end result is a remarkably versatile scaffold for the preparation of model heme adducts to mimic the heme iron coordination structure of native heme proteins. In this review, we first summarize the quantitative evidence for differential ligand binding affinities of the proximal and distal pockets of the H93G Mb cavity mutant that facilitates the preparation of mixed-ligand derivatives. Then we review our use of magnetic circular dichroism and electronic absorption spectroscopy to characterize nitrogen-, oxygen-, and sulfur-donor-ligated H93G Mb adducts with an emphasis on species not easily prepared by other heme model system approaches and those that serve as spectroscopic models for native heme proteins.

The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11

“…The lowest energy feature in the MCD spectra of MmpL3-E1 and MmpL11-E1 is a negative band at 638 nm. This feature is similar to the ligandto-metal charge transfer bands observed in ferric Mb at pH 6.8 and cyclohexylamine-ligated H93G Mb, suggesting that one of the high spin species present in MmpL3-E1 and MmpL11-E1 is a His-or Lys-ligated heme (44,45). The wavelength and sign of the broad positive feature centered at 594 nm is most similar to the positive ligand-to-metal charge transfer bands in three anionic oxygen-bound heme proteins as follows: alkaline H93G Mb (62); H93Y Mb (62); and C436S CYP2B4 (63).…”

“…The most intense feature in the MCD spectrum of both species is a derivative-shaped feature centered at 410 nm, which corresponds to a shoulder observed in the absorption spectrum of MmpL3-E1. The wavelength and intensity of this feature are consistent with the Soret band of a low spin heme species (44,45). Absorption spectra were acquired for both MmpL3-E1 and MmpL11-E1 at 20 K (data not shown) using the same samples and instrumental setup as described for MCD spectroscopy, and the Soret peak maxima were between 380 and 390 nm, consistent with the presence of a high spin heme species.…”

Electron Accepting Units of the Diheme Cytochrome c TsdA, a Bifunctional Thiosulfate Dehydrogenase/Tetrathionate Reductase

“…The fully oxidized diheme cytochrome AvTsdA contains His/Cys coordination in heme 1 and His/Lys in heme 2. His/Cys-ligated hemes are typically distinguished from other low-spin c-type hemes by having much lower reduction potentials and smaller changes in extinction coefficient associated with the Fe(III)/(II) couple (4,13). As a consequence, we propose that reduction of His/Cys-ligated heme 1 occurs reversibly between approximately Ϫ100 and Ϫ350 mV.…”

Hydrogen bond donation to the heme distal ligand of Staphylococcus aureus IsdG tunes the electronic structure

“…Circular dichroism (CD) spectroscopy has been employed more sparingly, but has proven to be an extremely sensitive probe of active site geometric structure [32,33]. Room temperature magnetic CD (MCD) spectroscopy has been extensively employed to "fingerprint" the oxidation, spin, and coordination state of heme proteins [34]. Additional electronic structure information is available when MCD data is acquired at cryogenic temperatures, in particular the ground state electron configuration can be determined [35].…”