Magnetic field effects as a result of the radical pair mechanism are unlikely in redox enzymes

Environmental exposure to electromagnetic fields is potentially carcinogenic. The radical pair mechanism is considered the most feasible mechanism of interaction between weak magnetic fields encountered in our environment and biochemical systems. Radicals are abundant in biology, both as free radicals and reaction intermediates in enzyme mechanisms. The catalytic cycles of some flavin-dependent enzymes are either known or potentially involve radical pairs. Here, we have investigated the magnetic field sensitivity of a number of flavoenzymes with important cellular roles. We also investigated the magnetic field sensitivity of a model system involving stepwise reduction of a flavin analogue by a nicotinamide analogue—a reaction known to proceed via a radical pair. Under the experimental conditions used, magnetic field sensitivity was not observed in the reaction kinetics from stopped-flow measurements in any of the systems studied. Although widely implicated in radical pair chemistry, we conclude that thermally driven, flavoenzyme-catalysed reactions are unlikely to be influenced by exposure to external magnetic fields

Reduction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M–1 s–1. In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s–1. At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is 51 s–1, whereas without 3-HB, the rate constant is 0.43 s–1 at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme–product (2,5-DHB) complex, with a rate constant of 45 ± 2 s–1. The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because Em0 for the enzyme–substrate complex is -179 ± 1 mV compared to an Em0 of -175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme–ligand complex to a fully activated form before flavin reduction takes plac

Elucidation of the biosynthesis of the methane catalyst coenzyme F430

Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(II)-thiolate intermediate. However, it is unclear how coenzyme F430 is synthesized from the common primogenitor uroporphyrinogen III, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F430 from sirohydrochlorin, termed CfbA–CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely