Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

Abstract

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH 2 -truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H 2 O 2 without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542-553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a fivecoordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O 2 ) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O-O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.✩ We dedicate this article to Professor Minor J. (Jud) Coon in recognition of his brilliant contributions and outstanding leadership to the field of cytochrome P450 over the past five decades. We wish to especially draw attention to his exceptional qualities as a role model, not just for how to do world-class science, but also for how to be a wonderfully supportive colleague and friend. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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

“…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). This suggests the presence of a second high spin (Tyr or Ser)-ligated heme.…”

A redox-mediated Kemp eliminase

“…It is well known that a cysteine-to-serine mutation30 in P450-BM3 at the axial Fe-binding position C400S eliminates monooxygenation activity but enhances the efficiency of Fe III -to-Fe II reduction by using NADPH as a reductant26. Therefore, we tested P450-BM3 variant C400S in the Kemp elimination of 1 .…”

Coordination modes of tyrosinate-ligated catalase-type heme enzymes: Magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp. paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states

“…We have recently demonstrated that the axial ligand mutant H93Y Mb, phenol-bound cavity mutant H93G Mb (which lacks the proximal His), and an axial ligand P450 mutant (C436S CYP2B4) all have very similar UV-Vis absorption and MCD spectra in the ferric state [27]. The results have led us to conclude that the latter two mutant heme proteins have an anionic oxygen atom donor ligated to the heme iron in a five-coordinate mode [27], as established for H93Y Mb [10], i.e., with an axial ligand being provided by tyrosinate for H93Y Mb, phenolate for H93G Mb and serinate for C436S CYP2B4.…”

“…The results have led us to conclude that the latter two mutant heme proteins have an anionic oxygen atom donor ligated to the heme iron in a five-coordinate mode [27], as established for H93Y Mb [10], i.e., with an axial ligand being provided by tyrosinate for H93Y Mb, phenolate for H93G Mb and serinate for C436S CYP2B4. To propose a basis (or bases) for the spectral differences (see Results, section 3.1) between this group of phenolate (or alkoxylate)-ligated (mutant) heme proteins and the aforementioned tyrosinate-ligated (native) heme enzymes (cAOS, MAP and BLC), we compare the active site structures of the latter three proteins with that of H93Y Mb in detail (Fig.…”

“…Thus, it may be reasonable to consider tyrosinate (Tyr-O − ) to be the axial heme iron ligand trans to bound CO for cAOS, MAP and BLC. On the other hand, neutral (protonated) Tyr (Tyr-OH) and water (H 2 O) have been postulated to be the axial ligand for Fe(II)-CO H93Y Mb [27] and H93G Mb [37], respectively. It has been shown that, in H93Y Mb (as well as H93C Mb), the negatively charged axial ligand (Tyr-O − or Cys-S − ) to the ferric heme iron cannot be retained upon reduction of the iron [38, 39].…”