抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

ion. The oxidative addition mechanism was originally proposed22i because of the lack of a strong rate dependence on polar factors and on the acidity of the medium. Later, however, the electrophilic substitution mechanism also was proposed. Recently, the oxidative addition mechanism was confirmed by investigations into the decomposition and protonolysis of alkylplatinum complexes, which are the reverse of alkane activation. There are two routes which operate in the decomposition of the dimethylplatinum(IV) complex Cs2Pt(CH3)2Cl4. The first route leads to chloride-induced reductive elimination and produces methyl chloride and methane. The second route leads to the formation of ethane. There is strong kinetic evidence that the ethane is produced by the decomposition of an ethylhydridoplatinum(IV) complex formed from the initial dimethylplatinum(IV) complex. In D2O-DCl, the ethane which is formed contains several D atoms and has practically the same multiple exchange parameter and distribution as does an ethane which has undergone platinum(II)-catalyzed H-D exchange with D2O. Moreover, ethyl chloride is formed competitively with H-D exchange in the presence of platinum(IV). From the principle of microscopic reversibility it follows that the same ethylhydridoplatinum(IV) complex is the intermediate in the reaction of ethane with platinum(II). Important results were obtained by Labinger and Bercaw62c in the investigation of the protonolysis mechanism of several alkylplatinum(II) complexes at low temperatures. These reactions are important because they could model the microscopic reverse of C-H activation by platinum(II) complexes. Alkylhydridoplatinum(IV) complexes were observed as intermediates in certain cases, such as when the complex (tmeda)Pt(CH2Ph)Cl or (tmeda)PtMe2 (tmeda ) N,N,N′,N′-tetramethylenediamine) was treated with HCl in CD2Cl2 or CD3OD, respectively. In some cases H-D exchange took place between the methyl groups on platinum and the, CD3OD prior to methane loss. On the basis of the kinetic results, a common mechanism was proposed to operate in all the reactions: (1) protonation of Pt(II) to generate an alkylhydridoplatinum(IV) intermediate, (2) dissociation of solvent or chloride to generate a cationic, fivecoordinate platinum(IV) species, (3) reductive C-H bond formation, producing a platinum(II) alkane σ-complex, and (4) loss of the alkane either through an associative or dissociative substitution pathway. These results implicate the presence of both alkane σ-complexes and alkylhydridoplatinum(IV) complexes as intermediates in the Pt(II)-induced C-H activation reactions. Thus, the first step in the alkane activation reaction is formation of a σ-complex with the alkane, which then undergoes oxidative addition to produce an alkylhydrido complex. Reversible interconversion of these intermediates, together with reversible deprotonation of the alkylhydridoplatinum(IV) complexes, leads to multiple H-D exchange

Activation of c-h bonds by metal complexes

The selective transformation of ubiquitous but inert C–H bonds to other functional groups has far-reaching practical implications, ranging from more efficient strategies for fine chemical synthesis to the replacement of current petrochemical feedstocks by less expensive and more readily available alkanes. The past twenty years have seen many examples of C–H bond activation at transition-metal centres, often under remarkably mild conditions and with high selectivity. Although profitable practical applications have not yet been developed, our understanding of how these organometallic reactions occur, and what their inherent advantages and limitations for practical alkane conversion are, has progressed considerably. In fact, the recent development of promising catalytic systems highlights the potential of organometallic chemistry for useful C–H bond activation strategies that will ultimately allow us to exploit Earth's alkane resources more efficiently and cleanly.

Understanding and exploiting C–H bond activation

ion phase that leads to an alkyl radical coordinated to the iron-hydroxo complex by a weak OH---C hydrogen bond, labeled as CI; (ii) an alkyl (or OH) rotation phase whereby the alkyl group achieves a favorable orientation for rebound; and (iii) a rebound phase that leads to C-O bond making and the ferric-alcohol complexes, 4,2P. The two profiles remain close in energy throughout the first two phases and then bifurcate. Whereas the HS state exhibits a significant barrier and a genuine TS for rebound, in the LS state, once the right orientation of the alkyl group is achieved, the LS rebound proceeds in a virtually barrier-free fashion to the alcohol. As such, alkane hydroxylation proceeds by TSR,70-72,120 in which the HS mechanism is truly stepwise with a finite lifetime for the radical intermediate, whereas the LS mechanism is effectively concerted with an ultrashort lifetime for the radical intermediate. Subsequent studies of ethane and camphor hydroxylation by the Yoshizawa group117,181-183 arrived at basically the same conclusion, that the mechanism is typified by TSR. The differences between the results of Shaik et al.130,173,177-180 and Yoshizawa et al.117,181-183 were rationalized recently71,72 and shown to arise owing to technical problems and the choice of the mercaptide ligand,117,181-183 which is a powerful electron donor and is too far from the representation of cysteine in the protein environment. The most recent study of camphor hydroxylation, which was done at a higher quality,117 converged to the picture reported by Shaik et al.130,173,177-180 and shows a stepwise HS process with a barrier of more than 3 kcal/mol for C-O bond formation by rebound of the camphoryl radical vis-à-vis an effectively concerted LS process for which this barrier is 0.7 kcal mol-1 and is the rotational barrier for reaching the rebound position. By referring to Figure 21, it is possible to rationalize the clock data of Newcomb in a simple manner. The apparent lifetimes are based on the assumption that there is a single state that leads to the reaction, such that the radical lifetime can be quantitated from the rate constant of free radical rearrangement and the ratio of rearranged to unrearranged alcohol product. However, in TSR, the rearranged (R) product is formed only/mainly on the HS surface, while the unrearranged (U) product is formed mainly on Figure 20. Formal descriptions of iron(III)-peroxo, iron(III)-hydroperoxo, and iron(V)-oxo species with indication of the negative charges. The roles “electrophile” or “nucleophile” are assigned according to the charge type. Reprinted with permission from ref 7. Copyright 2000 Springer-Verlag Heidelberg. 3964 Chemical Reviews, 2004, Vol. 104, No. 9 Meunier et al.

Mechanism of oxidation reactions catalyzed by cytochrome p450 enzymes.

Two decades have passed since the discovery in liver microsomes of a haemprotein that forms a reduced-CO complex with the absorptive maximum of the Soret at 450 nm (Klingenberg, 1958; Garfinkel, 1958) and the identification of this protein as a new cytochrome: pigment cytochrome, P-450 (Omura and Sato, 1962, 1964a). In the intervening years, the study of cytochrome P-450 dependent monoxygenases has expanded exponentially. From the first crude attempts to solubilise a P-450 (Omura and Sato, 1963, 1964b) to the determination of the primary, secondary, and tertiary structure of cytochrome P-450cam by amino acid sequencing (Haniu et al., 1982a,b) and x-ray crystallography (Poulos et al., 1984) our understanding of this unique family of proteins has been advancing on all fronts. Since, perhaps, the greatest understanding of the structure and mechanism of P-450s has come from concentrated study of P-450cam of the Pseudomonas putida camphor-5-exo-hydroxylase, this review will concentrate on findings with P-450cam; attention will be drawn to parallel and contrasting examples from other P-450s as appropriate.

Structure and Chemistry of Cytochrome P450

P450BM3 (CYP102A1), a fatty acid hydroxylase from Bacillus megaterium, has been extensively studied over a period of almost forty years. The enzyme has been redesigned to catalyse the oxidation of non-natural substrates as diverse as pharmaceuticals, terpenes and gaseous alkanes using a variety of engineering strategies. Crystal structures have provided a basis for several of the catalytic effects brought about by mutagenesis, while changes to reduction potentials, inter-domain electron transfer rates and catalytic parameters have yielded functional insights. Areas of active research interest include drug metabolite production, the development of process-scale techniques, unravelling general mechanistic aspects of P450 chemistry, methane oxidation, and improving selectivity control to allow the synthesis of fine chemicals. This review draws together the disparate research themes and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field.

P450BM3 (CYP102A1): connecting the dots

Carbon-hydrogen-transition metal bonds

Cytochrome P450 (CYP) enzymes are involved in activating the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) in mammals, but they are also utilized by microorganisms for the degradation of these hazardous environmental contaminants. Wild-type CYP102 (P450(BM-3)) from Bacillus megaterium has low activity for the oxidation of the PAHs phenanthrene, fluoranthene and pyrene. The double hydrophobic substitution R47L/Y51F at the entrance of the substrate access channel increased the PAH oxidation activity by up to 40-fold. Combining these mutations with the active site mutations F87A and A264G lead to order of magnitude increases in activity. Both these mutations increased the NADPH turnover rate, but the A264G mutation increased the coupling efficiency while the F87A mutation had dominant effects in product selectivity. Fast NADPH oxidation rates were observed (2250 min-1 for the R47L/Y51F/F87A mutant with phenanthrene) but the coupling efficiencies were relatively low (< 13%), resulting in a highest substrate oxidation rate of 110 min-1 for fluoranthene oxidation by the R47L/Y51F/A264G mutant. Mutation of M354 and L437 inside the substrate access channel reduced PAH oxidation activity. The PAHs were oxidized to a mixture of phenols and quinones. Notably mutants containing the A264G mutation showed some similarity to mammalian CYP enzymes in that some 9,10-phenanthrenequinone, the K-region oxidation product from phenanthrene, was formed. The results suggest that CYP102 mutants could be useful models for PAH oxidation by mammalian CYP enzymes, and also potentially for the preparation of novel PAH bioremediation systems.

Protein engineering of Bacillus megaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons.

The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)−1 min−1, with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min−1) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3.

Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3

Carbon-hydrogen bonds, especially those of saturated (s$) carbon centres, are normally regarded as being chemically inert. Generally, the C-H group is not thought of as a potential ligand which can have a structural role or play an energetically significant part in ground states or in reaction intermediates. In this article we review recent observations which show that there are in fact many circumstances in which a carbon-hydrogen group will interact with a transition metal centre with formation of a two-electron three-centre bond and that the extent of the interaction is such as to have a marked effect on the molecular and electronic structure and hence reactivity of the molecule. We propose the term “agostic” which will be used to discuss the various manifestations of covalent interactions between carbon-hydrogen groups and transition metal centres in organometallic compounds l . The word agostic will be used to refer specifically to situations in which a hydrogen atom is covalently bonded simultaneously to both a carbon atom and to a transition metal atom. The agostic C-H-M bond is similar to the familiar and long known bridging hydrogen systems which occur in B-H-B, M-H-M, and B-H-M groups. The C-H-M bonds are a recent observation but as will be discussed below, they are probably very much more common than hitherto suspected. Since carbon-hydrogen bonds are a ubiquitous feature of organometallic compounds we have found it useful to define

Luet-Lok Wong

Papers

P450BM3 (CYP102A1): connecting the dots

Carbon-hydrogen-transition metal bonds

Protein engineering of Bacillus megaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons.

Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3

Carbon-Hydrogen-Transition Metal Bonds