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CPR (NADPH–cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs. [ABSTRACT FROM AUTHOR]

ROS (reactive oxygen species) are considered to be a major cause of cellular oxidative stress, linked to neuromuscular diseases and aging. Complex I (NADH:ubiquinone oxidoreductase) is one of the main contributors to superoxide production by mitochondria, and knowledge of its mechanism of O2 reduction is required for the formulation of causative connections between complex I defects and pathological effects. There is evidence for two distinct (but not mutually exclusive) sites of O2 reduction by complex I. Studies of the isolated enzyme largely support the participation of the reduced flavin mononucleotide in the active site for NADH oxidation, and this mechanism is supported in mitochondria by correlations between the NAD(P)+ potential and O2 reduction. In addition, studies of intact mitochondria or submitochondrial particles have suggested a mechanism involving the quinone-binding site, supported by observations during reverse electron transport and the use of 'Q-site' inhibitors. Here, we discuss extant data and models for O2 reduction by complex I. We compare results from the isolated enzyme with results from intact mitochondria, aiming to identify similarities and differences between them and progress towards combining them to form a single, unified picture. [ABSTRACT FROM AUTHOR]

BVR-B (biliverdin-IXβ reductase) also known as FR (flavin reductase) is a promiscuous enzyme catalysing the pyridine-nucleotide-dependent reduction of a variety of flavins, biliverdins, PQQ (pyrroloquinoline quinone) and ferric ion. Mechanistically it is a good model for BVR-A (biliverdin-IXα reductase), a potential pharmacological target for neonatal jaundice and also a potential target for adjunct therapy to maintain protective levels of biliverdin-IXα during organ transplantation. In a commentary on the structure of BVR-B it was noted that one outstanding issue remained: whether the mechanism was a concerted hydride transfer followed by protonation of a pyrrolic anion or protonation of the pyrrole followed by hydride transfer. In the present study we have attempted to address this question using QM/MM (quantum mechanics/molecular mechanics) calculations. QM/MM potential energy surfaces show that the lowest energy pathway proceeds with a positively charged pyrrole intermediate via two transition states. These initial calculations were performed with His153 as the source of the proton. However site-directed mutagenesis studies with both the H153A and the H153N mutant reveal that His153 is not required for catalytic activity. We have repeated the calculation with a solvent hydroxonium donor and obtain a similar energy landscape indicating that protonation of the pyrrole is the most likely first step followed by hydride transfer and that the required proton may come from bulk solvent. The implications of the present study for the design of inhibitors of BVR-A are discussed. [ABSTRACT FROM AUTHOR]