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Paired Spectroscopic and Crystallographic Studies of Proteases.

ABSTRACT: The active sites of subtilisin and trypsin have been studied by paired IR spectroscopic and X-ray crystallographic studies. The active site serines of the proteases were reacted with 4-cyanobenzenesulfonyl fluoride (CBSF), an inhibitor that contains a nitrile vibrational reporter. The nitrile stretch vibration of the water-soluble inhibitor model, potassium 4-cyanobenzenesulfonate (KCBSO), and the inhibitor were calibrated by IR solvent studies in H₂O/DMSO and the frequency-temperature line-slope (FTLS) method in H₂O and THF. The inhibitor complexes were examined by FTLS and the slopes of the best fit lines for subtilisin-CBS and trypsin-CBS in aqueous buffer were both measured to be -3.5×10^-2 cm^-1/°C. These slopes were intermediate in value between that of KCBSO in aqueous buffer and CBSF in THF, which suggests that the active-site nitriles in both proteases are mostly solvated. The X-ray crystal structures of the subtilisin-CBS and trypsin-CBS complexes were solved at 1.27 and 1.32 Å, respectively. The inhibitor was modelled in two conformations in subtilisin-CBS and in one conformation in the trypsin-CBS. The crystallographic data support the FTLS data that the active-site nitrile groups are mostly solvated and participate in hydrogen bonds with water molecules. The combination of IR spectroscopy utilizing vibrational reporters paired with X-ray crystallography provides a powerful approach to studying protein structure.

SUBMITTER: Luo M

PROVIDER: S-EPMC8221577 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the ?-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the ?- and ?-meso carbons, whereas the spin density distribution may favor selective oxygenation of the ?-meso carbon.

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Paired Spectroscopic and Crystallographic Studies of Proteases.

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