Ying-Wu Lin

Regulating the coordination state of a heme protein by a designed distal hydrogen-bonding network.

Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins.

A Novel Tyrosine-Heme C-O Covalent Linkage in F43Y Myoglobin: A New Post-translational Modification of Heme Proteins

Heme post‐translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine–heme covalent Cuf8ffO bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4‐vinyl group. This highlights the diverse chemistry of heme post‐translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently‐bound heme proteins.

Structural and nitrite reductase activity comparisons of myoglobins with one to three distal histidines.

Although with a distinct heme active site, both myoglobin (Mb) and cytochrome c oxidase (CcO) were found to function as a nitrite reductase (NIR) under hypoxic conditions. On the other hand, Mb was rationally designed to mimic native CcO by introduction of two distal histidines, i.e., L29H/F43H mutant, where His29, His43 and native His64 formed a metal-binding site. To probe the role of distal histidines in regulating the NIR activity of Mb, we herein designed a single mutant of L29H Mb that contains two distal histidines and solved its X-ray crystal structure, then we made both structural and NIR activity comparisons of Mbs with one to three distal histidines. It was shown that introduction of one or two additional distal histidines in Mb leads to the formation of a distal hydrogen network, which slightly alters the conformation of heme active site and inhibits the NIR activity. Meanwhile, the reductase activity of both L29H Mb and L29H/F43H Mb are regulated by metal ions such as Cu(II) or Zn(II) binding to the distal histidines, i.e., Cu(II) enhances the reactivity while Zn(II) inhibits the reactivity. These findings provide valuable insights into the structure and function relationship for both Mb and CcO functioning as a NIR in biological system.

Distinct mechanisms for DNA cleavage by myoglobin with a designed heme active center.

Heme proteins perform diverse biological functions, of which myoglobin (Mb) is a representative protein. In this study, the O2 carrier Mb was shown to cleave double stranded DNA upon aerobic dithiothreitol-induced reduction, which is fine-tuned by an additional distal histidine, His29 or His43, engineered in the heme active center. Spectroscopic (UV-vis and EPR) and inhibition studies suggested that free radicals including singlet oxygen and hydroxyl radical are responsible for efficient DNA cleavage via an oxidative cleavage mechanism. On the other hand, L29E Mb, with a distinct heme active center involving three water molecules in the met form, was found to exhibit an excellent DNA cleavage activity that was not depending on O2. Inhibition and ligation studies demonstrated for the first time that L29E Mb cleaves double stranded DNA into both the nicked circular and linear forms via a hydrolytic cleavage mechanism, which resembles native endonucleases. This study provides valuable insights into the distinct mechanisms for DNA cleavage by heme proteins, and lays down a base for creating artificial DNA endonucleases by rational design of heme proteins. Moreover, this study suggests that the diverse functions of heme proteins can be fine-tuned by rational design of the heme active center with a hydrogen-bonding network.

How a novel tyrosine–heme cross-link fine-tunes the structure and functions of heme proteins: a direct comparitive study of L29H/F43Y myoglobin

A heme-protein cross-link is a key post-translational modification (PTM) of heme proteins. Meanwhile, the structural and functional consequences of heme-protein cross-links are not fully understood, due to limited studies on a direct comparison of the same protein with and without the cross-link. A Tyr-heme cross-link with a C-O bond is a newly discovered PTM of heme proteins, and is spontaneously formed in F43Y myoglobin (Mb) between the Tyr hydroxyl group and the heme 4-vinyl group in vivo. In this study, we found that with an additional distal His29 introduced in the heme pocket, the double mutant L29H/F43Y Mb can form two distinct forms under different protein purification conditions, with and without a novel Tyr-heme cross-link. By solving the X-ray structures of both forms of L29H/F43Y Mb and performing spectroscopic studies, we made a direct structural and functional comparison in the same protein scaffold. It revealed that the Tyr-heme cross-link regulates the heme distal hydrogen-bonding network, and fine-tunes not only the spectroscopic and ligand binding properties, but also the protein reactivity. Moreover, the formation of the Tyr-heme cross-link in the double mutant L29H/F43Y Mb was investigated in vitro. This study addressed the key issue of how Tyr-heme cross-link fine-tunes the structure and functions of the heme protein, and provided a plausible mechanism for the formation of the newly discovered Tyr-heme cross-link.

Regulating the nitrite reductase activity of myoglobin by redesigning the heme active center

Heme proteins perform diverse functions in living systems, of which nitrite reductase (NIR) activity receives much attention recently. In this study, to better understand the structural elements responsible for the NIR activity, we used myoglobin (Mb) as a model heme protein and redesigned the heme active center, by introducing one or two distal histidines, and by creating a channel to the heme center with removal of the native distal His64 gate (His to Ala mutation). UV-Vis kinetic studies, combined with EPR studies, showed that a single distal histidine with a suitable position to the heme iron, i.e., His43, is crucial for nitrite (NO2(-)) to nitric oxide (NO) reduction. Moreover, creation of a water channel to the heme center significantly enhanced the NIR activity compared to the corresponding mutant without the channel. In addition, X-ray crystallographic studies of F43H/H64Axa0Mb and its complexes with NO2(-) or NO revealed a unique hydrogen-bonding network in the heme active center, as well as unique substrate and product binding models, providing valuable structural information for the enhanced NIR activity. These findings enriched our understanding of the structure and NIR activity relationship of heme proteins. The approach of creating a channel in this study is also useful for rational design of other functional heme proteins.

Mimicking a Natural Enzyme System: Cytochrome c Oxidase-Like Activity of Cu2O Nanoparticles by Receiving Electrons from Cytochrome c

Inorganic nanomaterials-based artificial enzymes (nanozymes) have received considerable attention over the past years. However, the substrates studied for nanozymes have so far been limited to small organic molecules. The catalytic oxidation of biomacromolecules, such as proteins, by nanozymes has not yet been reported to date. In this study, we report that cuprous oxide nanoparticles (Cu2O NPs) possess cytochrome c oxidase (CcO)-like activity and catalyze the oxidation of cytochrome c (Cyt c), converting it from the ferrous state to the ferric state under atmospheric oxygen conditions. Furthermore, the CcO-like activity of Cu2O NPs is pH- and size-dependent. The lower the solution pH and the smaller the particle size, the higher the CcO-like activity. The artificial Cyt c-Cu2O NPs system closely mimics the native Cyt c-CcO enzyme system, which opens new vistas in enzyme construction and potential applications.

An intramolecular disulfide bond designed in myoglobin fine-tunes both protein structure and peroxidase activity

Disulfide bond plays crucial roles in stabilization of protein structure and in fine-tuning protein functions. To explore an approach for rational heme protein design, we herein rationally introduced a pair of cysteines (F46C/M55C) into the scaffold of myoglobin (Mb), mimicking those in native neuroglobin. Molecular modeling suggested that it is possible for Cys46 and Cys55 to form an intramolecular disulfide bond, which was confirmed experimentally by ESI-MS analysis, DTNB reaction and CD spectrum. Moreover, it was shown that the spontaneously formed disulfide bond of Cys46-Cys55 fine-tunes not only the heme active site structure, but also the protein functions. The substitution of Phe46 with Ser46 in F46S Mb destabilizes the protein while facilitates H2O2 activation. Remarkably, the formation of an intramolecular disulfide bond of Cys46-Cys55 in F46C/M55C Mb improves the protein stability and regulates the heme site to be more favorable for substrate binding, resulting in enhanced peroxidase activity. This study provides valuable information of structure-function relationship for heme proteins regulated by an intramolecular disulfide bond, and also suggests that construction of such a covalent bond is useful for design of functional heme proteins.

Structure and function of heme proteins regulated by diverse post-translational modifications

Heme proteins are crucial for biological systems by performing diverse functions. Nature has evolved diverse approaches to fine-tune the structure and function of heme proteins, of which post-translational modification (PTM) is a primary method. As reviewed herein, a multitude of PTMs have been discovered for heme proteins in the last several decades, including heme-protein cross-links with heme side chains (Cys-heme, Tyr-heme and Asp/Glu-heme, etc) or porphyrin ring (Lys-heme and Tyr-heme, etc), heme modifications (sulfheme and nitriheme, etc), amino acids cross-links between two or among multiple residues (Cys-Cys, Tyr-His, Tyr-Cys, Met-Tyr-Trp, etc), and amino acids modifications by oxidation, nitration, phosphorylation and glycation, etc. With the development of research methods and advances in research techniques, deep insights have been obtained for the formation mechanisms of PTMs, as well as their effects on the structure and function of heme proteins. Moreover, some positive PTMs have been successfully applied to create artificial heme proteins with advanced functions, whereas some negative PTMs have been regulated by rational design of inhibitors. The tremendous progress, together with those ongoing, will make it possible to rationally control the diverse PTMs of heme proteins, especially those associated with human diseases, toward our desired goals for a better life.

Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin

Abstract Rational protein design has been proven to be a powerful tool for creating functional artificial proteins. Although many artificial metalloproteins with a single active site have been successfully created, those with dual active sites in a single protein scaffold are still relatively rare. In this study, we rationally designed dual active sites in a single heme protein scaffold, myoglobin (Mb), by retaining the native heme site and creating a copper‐binding site remotely through a single mutation of Arg118 to His or Met. Isothermal titration calorimetry (ITC) and electron paramagnetic resonance (EPR) studies confirmed that a copper‐binding site of [3‐His] or [2‐His‐1‐Met] motif was successfully created in the single mutant of R118H Mb and R118M Mb, respectively. UV/Vis kinetic spectroscopy and EPR studies further revealed that both the heme site and the designed copper site exhibited nitrite reductase activity. This study presents a new example for rational protein design with multiple active sites in a single protein scaffold, which also lays the groundwork for further investigation of the structure and function relationship of heme/non‐heme proteins.