Cytochrome P450s (P450s) 2 are a superfamily of b-type hemoproteins responsible for the metabolism of a wide variety of exogenous compounds such as drugs and carcinogens, and endogenous compounds such as prostaglandins and steroids (1). P450 reactions require input of two electrons (supplemental Fig. S1) (1, 2). The efficiency of electron transfer is one of the key determinants of the reaction kinetics. In microsomal systems, NADPH is the ultimate source of the two electrons, and NADPH-dependent cytochrome P450 oxidoreductase (P450 reductase) together with cytochrome b 5 (b 5 ) facilitates the electron transfer. Knowledge of the interactions between P450s and their redox partners is fundamental to a complete understanding of the mechanisms of P450 reactions.The interactions between P450s and b 5 have drawn much attention because of variable effects b 5 has on different P450 isoforms and P450 reactions. It has been shown that b 5 may stimulate, inhibit or have no effects on P450-catalyzed reactions depending on the particular isoform of P450 and the substrate of the reaction (3-5). However, there is no consensus on whether b 5 transfers electrons to P450, or causes an allosteric effect on P450, or whether both mechanisms are simultaneously operative (6 -10). In addition, the mechanism of substrate-and P450 isoform dependency is unknown (5, 11).CYP2E1 is a P450 isoform whose reactions are highly stimulated in the presence of b 5 . For example, we previously found that b 5 stimulates CYP2E1-catalyzed oxidation of acetaminophen to its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), by 25-fold (12). For other CYP2E1-catalyzed reactions, such as aniline p-hydroxylation and 7-ethoxycoumarin O-deethylation, b 5 stimulates the reactions by 270-fold and 67-fold, respectively (5, 11). In contrast to its stimulating effects on CYP3A4, CYP3A5, CYP2C19, CYP2B6, and CYP2C8, apo-b 5 is unable to replace holo-b 5 in stimulating CYP2E1-catalyzed reactions (11,12). The requirement for the heme group increases the probability that b 5 stimulates CYP2E1-catalyzed reactions by facilitating electron transfer rather than by only causing a positive allosteric effect. Because intermolecular complex formation immediately precedes electron trans-* This work was supported by National Institutes of Health Grant GM32165 (tp S. D. N.), the UW NIEHS-sponsored Center for Ecogenetics and Environmental Health Grant P30ES07033, an NCRR high end instrumentation award 1S10RR17262-01 (to D. R. G.), and the WWAMI RCE for Biodefense and Emerging Infectious Diseases 1U54AI57141-01 (to D. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To facilitate structural analysis of proteins and protein-protein interactions, we developed Pro-CrossLink, a suite of software tools consisting of three programs (Figure 1), DetectShift, IdentifyXLink, and AssignXLink. DetectShift was developed to detect ions of cross-linked peptide pairs in a mixture of 18O-labeled peptides obtained from protein proteolytic digests. The selected candidate ions of cross-linked peptide pairs subsequently undergo tandem mass spectrometric (MS/MS) analysis for sequence determination. Based on the masses of candidate ions as well as y- and b-type ions in the tandem mass spectra, IdentifyXLink assigns the candidate ions to cross-linked peptide pairs. For an identified cross-linked peptide pair, AssignXLink generates an extensive fragment ion list, including a-, b-, c-type, x-, y-, z-type, internal, and immonium ions with associated common losses of H2O, NH3, CO, and CO2, and facilitates a precise location of the cross-linked residues. Pro-CrossLink is automated, highly configurable by the user, and applicable to many studies that map low-resolution protein structures and molecular interfaces in protein complexes.

Cytochrome b5 (cyt b5) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached on the underlying mechanism of cyt b5 modulation of CYP catalysis. Both cyt b5 and apo b5, are reported to stimulate the activity of several P450 isoforms. In the present study, the surface interactions of both holo and apo b5 with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the interaction models of holo/apo b5 with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b5 and apo b5 were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B’ loop of CYP3A4, a substrate recognition site (SRS). Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127 and Lys421) are of functional importance. Mutation of these residues reduced or abolished cyt b5 binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b5 and/or cytochrome P450 reductase (CPR) was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that the apo cyt b5 can dock with CYP3A4 in a manner analogous to holo cyt b5 so electron transfer from cyt b5 is not required for its effects.

Injudicious or inappropriate use of antibiotics has led to the prevalence of drug-resistant bacteria, posing a huge menace to global health. Here, a selfassembled aggregation-induced emission (AIE) nanosphere (AIE-PEG 1000 NPs) that simultaneously possesses near-infrared region II (NIR-II) fluorescence emissive, photothermal, and photodynamic properties is prepared using a multifunctional AIE luminogen (AIE-4COOH). The AIE-PEG 1000 NPs were encapsulated with teicoplanin (Tei) and ammonium bicarbonate (AB) into lipid nanovesicles to form a laseractivated "nanobomb" (AIE-Tei@AB NVs) for the multimodal theranostics of drugresistant bacterial infections. In vivo experiments validate that the "nanobomb" enables high-performance NIR-II fluorescence, infrared thermal, and ultrasound (AB decomposition during the photothermal process to produce numerous CO 2 /NH 3 bubbles, which is an efficient ultrasound contrast agent) imaging of multidrug-resistant bacteria-infected foci after intravenous administration of AIE-Tei@AB NVs followed by 660 nm laser stimulation. The highly efficient photothermal and photodynamic features of AIE-Tei@AB NVs, combined with the excellent pharmacological property of rapidly released Tei during bubble generation and NV disintegration, collectively promote broad-spectrum eradication of three clinically isolated multidrugresistant bacteria strains and rapid healing of infected wounds. This multimodal imaging-guided synergistic therapeutic strategy can be extended for the theranostics of superbugs.