Following formation PF-2341066 of the Fe(II)–CO complex, a new species is formed over 1–2 h, absorbing at about 420 nm, the so-called P420 form, which arises by protonation of the proximal cysteine thiolate ligand of the heme iron (Perera et al., 2003; Dunford et al., 2007). CO-binding assays were also performed with each P450 and the primary electron transfer proteins ecoFdR and one of spinFd, balFd-V, balFd-VII or ecoFld. These assays monitor the ability of each donor to transfer one electron to the P450 heme Fe(III), followed by binding of CO to the Fe(II) form. Although the proteins tested have diverse species origins (spinach, E. coli and A. balhimycina) and cofactor types ([2Fe–2S], FMN, [3Fe–4S]), all four were able to reduce

the Fe(III) heme of vanOxyB, albeit to different extents. The plant spinFd rapidly (<6 min) led to up to 75% conversion (relative to dithionite) to the vanOxyB P450-form, whereas with ecoFld, a maximum of 40% conversion was attained only after 40 min. A rapid formation of the P450 from vanOxyB (<10 min) was also observed using either Fd from A. balhimycina, with 60% conversion with balFd-V and 45% with balFd-VII. The OxyB enzymes from the balhimycin and vancomycin pathways share 88% sequence identity. Nevertheless, some differences were observed between these enzymes in their abilities to accept electrons from the four primary redox partners. Thus, both balOxyB and vanOxyB are rapidly (<5–6 min)

and efficiently (80%) converted into the P450 form by spinFd, but the emergence of the P450 form with balFd-V and balFd-VII was slower and reached at best 40% (balFd-V) and 20% (balFd-VII) of SB-3CT the response seen with sodium

Natural Product Library supplier dithionite. With balOxyB, essentially no reduction was observed using ecoFld. These CO-binding assays do not indicate whether or not a second electron transfer can occur to the heme as required in the full P450 catalytic cycle. For example, spinFd can donate the first electron to camphor-bound P450cam, but allows no hydroxylation of substrate (Lipscomb et al., 1976). To address this point, assays were also carried out with peptide substrates of OxyB. The assay for the catalytic activity of OxyB is that described in an earlier work (Zerbe et al., 2004; Woithe et al., 2007, 2008; Geib et al., 2008). The substrates used are the model hexa- and heptapeptides 1 and 2 (Fig. 1), which are closely related to the expected intermediates occurring during glycopeptide biosynthesis. Each peptide is covalently linked as a C-terminal thioester to an isolated recombinant PCP domain from the seventh module of the vancomycin NRPS. For ease of synthesis (Li & Robinson, 2005), these model peptides contain tyrosine at positions-2 and -6, rather than β-hydroxy-meta-chlorotyrosine (see Fig. 1). Standard conditions were used for all assays, so that a comparison of turnover efficiencies could be obtained from the extent of linear peptide conversion into the corresponding monocyclic product.