Measurement of Heme Ruffling Changes in MhuD Using UV–vis Spectroscopy

Graves, Amanda B.; Graves, Max T.; Liptak, Matthew D.

doi:10.1021/acs.jpcb.6b01497

Cited by 48 publications

(149 citation statements)

References 48 publications

(145 reference statements)

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“…Whereas the mean out-of-plane deviation for TPFPP diphenylcarbene was 1.03 Å (SI Appendix, Table S2), it is smaller for carbene-bound Rma TDE (0.6 Å; the deviation for Rma TDE alone is 0.7 Å). The difference in distortion between both Rma TDE structures is small, and both values are within the range reported for c-type cytochrome proteins (0.3-1.2 Å) (30). Ruffling is known to be the main distortion in these proteins and is thought to be induced by covalent attachment of the heme to the protein (31).…”

Section: Significancesupporting

confidence: 81%

Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase

Lewis

Garcia‐Borràs

Chalkley

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

106

149

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Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable () cytochrome High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in cytochrome has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.

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Section: Significancesupporting

confidence: 81%

Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase

Lewis

Garcia‐Borràs

Chalkley

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

106

149

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“…While the changes in the ν 10 marker band are broadly consistent with the changes in the NMR and EPR data, with a lower energy observed for more ruffled protein variants, the changes in this band are on the order of only a few cm −1 , while NMR and EPR spectra show significantly greater changes. We also note that UV-Vis absorption data also have been implemented as a probe of ruffling, but large changes in ruffling are required to see any shift in absorption bands [62].…”

Section: Spectroscopic Probes Of Heme Rufflingmentioning

confidence: 99%

Influence of heme c attachment on heme conformation and potential

et al. 2018

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Heme c is characterized by its covalent attachment to a polypeptide. The attachment is typically to a CXXCH motif in which the two Cys form thioether bonds with the heme, "X" can be any amino acid other than Cys, and the His serves as a heme axial ligand. Some cytochromes c, however, contain heme attachment motifs with three or four intervening residues in a CXCH or CXCH motif. Here, the impacts of these variations in the heme attachment motif on heme ruffling and electronic structure are investigated by spectroscopically characterizing CXCH and CXCH variants of Hydrogenobacter thermophilus cytochrome c. In addition, a novel CXCH variant is studied. H andC NMR, EPR, and resonance Raman spectra of the protein variants are analyzed to deduce the extent of ruffling using previously reported relationships between these spectral data and heme ruffling. In addition, the reduction potentials of these protein variants are measured using protein film voltammetry. The CXCH and CXCH variants are found to have enhanced heme ruffling and lower reduction potentials. Implications of these results for the use of these noncanonical motifs in nature, and for the engineering of novel heme peptide structures, are discussed.

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“…A prominent example of a protein that hosts a dynamic haem is Mycobacterium tuberculosis MhuD. [10][11][12] MhuD is a non-canonical haem oxygenase (HO) whose haem substrate undergoes a dynamic ruffling deformation, but it has not yet been ascertained whether the substrate dynamics have a functional role.…”

Section: Introductionmentioning

confidence: 99%

“…Magnetic measurements have shown that the large ruffling deformation observed in the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-haem-CN, PDB ID 4NL5) arises from a dynamic haem substrate that is present in solution as an equilibrium mixture of planar and ruffled haem. [10][11][12] One explanation for the presence of two haem conformations in MhuD is that it is a consequence of the fact that at least four polypeptide conformational states exist. 10,13,14 Alternatively, from a haem electronic structure viewpoint, the ruffled conformation can be attributed to a hidden pseudo Jahn-Teller distortion arising from vibronic coupling of the 2 B2g and 2 A2u excited states of the planar species by a b1u vibration.…”

Section: Introductionmentioning

confidence: 99%

“…This article describes a mechanistic study of MhuDcatalysed haem oxygenation carried out using UV/Vis absorption (Abs) spectroscopy and mass spectrometry (MS). Forms of MhuD with increased populations of the ruffled and planar substrate conformations were prepared by introducing the F23W and W66F substitutions, 11,12 respectively. The haem degradation products of WT, F23W, and W66F MhuD were identified using UV/Vis Abs spectroscopy and in proteo MS. 22 In addition, tandem MS was employed to determine the product isomer(s) for each reaction.…”

Section: Introductionmentioning

confidence: 99%

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A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

Thakuri¹,

O'Rourke²,

Graves³

et al. 2021

Preprint

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The non-canoncial haem oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a haem substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between haem substrate dynamics and MhuD-catalysed haem degradation resulting in a revised enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionisation mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favouring population of the ruffled haem conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyhaem intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favoured the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of haem monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyhaem dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid haem monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered haem monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyhaem. Altogether, these data yielded a revised enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into haem yielding mycobilin.<br>

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Measurement of Heme Ruffling Changes in MhuD Using UV–vis Spectroscopy

Cited by 48 publications

References 48 publications

Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase

Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase

Influence of heme c attachment on heme conformation and potential

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

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