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The crystal structure of horseradish peroxidase isozyme C (HRPC) has been solved to 2.15 A resolution. An important feature unique to the class III peroxidases is a long insertion, 34 residues in HRPC, between helices F and G. This region, which defines part of the substrate access channel, is not present in the core conserved fold typical of peroxidases from classes I and II. Comparison of HRPC and peanut peroxidase (PNP), the only other class III (higher plant) peroxidase for which an X-ray structure has been completed, reveals that the structure in this region is highly variable even within class III. For peroxidases of the HRPC type, characterized by a larger FG insertion (seven residues relative to PNP) and a shorter F' helix, we have identified the key residue involved in direct interactions with aromatic donor molecules. HRPC is unique in having a ring of three peripheral Phe residues, 142, 68 and 179. These guard the entrance to the exposed haem edge. We predict that this aromatic region is important for the ability of HRPC to bind aromatic substrates.

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and are difficult to use2. Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed3,4. MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix.

The crystal structure of Pseudomonas putida cytochrome P-450cam in the substrate-free form has been refined at 2.20-A resolution and compared to the substrate-bound form of the enzyme. In the absence of the substrate camphor, the P-450cam heme iron atom is hexacoordinate with the sulfur atom of Cys-357 providing one axial heme ligand and a water molecule or hydroxide ion providing the other axial ligand. A network of hydrogen-bonded solvent molecules occupies the substrate pocket in addition to the iron-linked aqua ligand. When a camphor molecule binds, the active site waters including the aqua ligand are displaced, resulting in a pentacoordinate high-spin heme iron atom. Analysis of the Fno camphor - F camphor difference Fourier and a quantitative comparison of the two refined structures reveal that no detectable conformational change results from camphor binding other than a small repositioning of a phenylalanine side chain that contacts the camphor molecule. However, large decreases in the mean temperature factors of three separate segments of the protein centered on Tyr-96, Thr-185, and Asp-251 result from camphor binding. This indicates that camphor binding decreases the flexibility in these three regions of the P-450cam molecule without altering the mean position of the atoms involved.

The crystal structure of the complex between the heme-and FMN-binding domains of bacterial cytochrome P450BM-3, a prototype for the complex between eukaryotic microsomal P450s and P450 reductase, has been determined at 2.03 Å resolution. The f lavodoxin-like f lavin domain is positioned at the proximal face of the heme domain with the FMN 4.0 and 18.4 Å from the peptide that precedes the heme-binding loop and the heme iron, respectively. The hemebinding peptide represents the most efficient and coupled through-bond electron pathway to the heme iron. Substantial differences between the FMN-binding domains of P450BM-3 and microsomal P450 reductase, observed around the f lavinbinding sites, are responsible for different redox properties of the FMN, which, in turn, control electron f low to the P450.Cytochromes P450, a gene superfamily of heme proteins found in all eukaryotes, most prokaryotes, and Archaea (1), catalyze the monooxygenation of a wide variety of organic molecules. P450 reactions of biological significance include steroid biogenesis, drug metabolism, procarcinogen activation, xenobiotic detoxification, and fatty acid metabolism (2, 3). Electron transfer from a redox partner to the P450 is a key step in the P450 catalytic cycle. Bacterial and mitochondrial P450s receive electrons from a small soluble iron-sulfur protein, whereas the redox partner for mammalian microsomal enzymes is an FAD͞FMN-dependent NADPH-cytochrome P450 oxidoreductase (CPR). In CPR, FAD serves as an electron acceptor from NADPH, whereas the FMN moiety interacts with and reduces the P450. The problem of redox partner recognition and mechanism of electron transfer has been one of the most important and intriguing in the area of P450 research, in particular, and in biological electron-transfer reactions, in general. The involvement of both electrostatic and hydrophobic forces in protein-protein interactions between P450s and their redox partners has been demonstrated (4-9). Although the structures of four bacterial P450s, putidaredoxin, adrenodoxin, and a soluble form of rat CPR are known (10-16), the questions of where and how P450s interact with electron donors and the precise nature of the electron-transfer mechanism remain to be answered.Flavocytochrome P450BM-3 (119 kDa), a self-sufficient fatty acid monooxygenase from Bacillus megaterium (17, 18), consists of a heme-(BMP) and FMN͞FAD-containing reductase domains linked together on a single polypeptide. Being a soluble multidomain electron-transfer protein, this enzyme represents an excellent model system for studying structure͞ function relationships in P450s and the mechanism of electron transfer. Expression of the individual domains and subdomains of P450BM-3 significantly facilitated studies on the mechanism of domain-domain interaction and interdomain electron transfer (19 -25). The heme͞FMN-containing domain of P450BM-3 (BMP͞FMN, missing the FAD domain) was found to be the simplest model to follow the FMN to heme intramolecular electron transfer (24, 25). Here we rep...

Nitric oxide, a key signaling molecule, is produced by a family of enzymes collectively called nitric oxide synthases (NOS). Here, we report the crystal structure of the heme domain of endothelial NOS in tetrahydrobiopterin (H4B)-free and -bound forms at 1.95 A and 1.9 A resolution, respectively. In both structures a zinc ion is tetrahedrally coordinated to pairs of symmetry-related cysteine residues at the dimer interface. The phylogenetically conserved Cys-(X)4-Cys motif and its strategic location establish a structural role for the metal center in maintaining the integrity of the H4B-binding site. The unexpected recognition of the substrate, L-arginine, at the H4B site indicates that this site is poised to stabilize a positively charged pterin ring and suggests a model involving a cationic pterin radical in the catalytic cycle.

The substrate-bound structures of two cytochrome P450s, P450cam and P450eryF, are known. While these structures reveal important features that control substrate specificity, the problem of how conformational changes allow for substrate entry and product release remains unsolved. The structure of the haem domain of the bacterial fatty acid hydroxylase, P450BM-3, previously was solved in the substrate-free form. Unlike the substrate-bound P450cam and P450eryF structures, the substrate access channel is open in substrate-free P450BM-3. Here we present the X-ray structure of P450BM-3 at 2.7 A bound with a fatty acid substrate, palmitoleic acid. A comparison of the substrate-bound and -free forms reveals major conformational differences and provides the first detailed picture of substrate-induced conformational changes in a P450.