Nuclear magnetic resonance of paramagnetic metalloporphyrins

The present article reviews and discusses proton magnetic resonance studies on metalloporphyrins which provide good models for heme proteins in their physical and chemical properties. Emphasis is given on the discussion of the 1HNMR work done in our laboratory

Electronic structure of spin-mixed iron(III) porphyrins: a proton magnetic resonance study

The proton magnetic resonance studies on the perchlorato iron(III) porphyrins in solution have been described. The isotropic proton shifts in these complexes show anomalous temperature dependence, consistent with its unusual properties in solid state. The NMR data have been analysed on the basis of a crystal field theory which includes lower asymmetric field and spin-orbit interaction. The analysis brings out that the ground state of the ferric ion in these porphyrin complexes exhibits the novel spin-mixed behaviour with spin-mixing between S = 3/2 and S = 5/2. The ground state is predominantly a spin quartet with the spin sextet being a very close lying excited state. Such a spin situation and spin-mixing have been speculated for the ferric ion in some ferricytochromec'. The present paper also highlights that the isotropic proton shift is very sensitive to the electronic structure of the metal ion and hence can be used to determine the electronic structure of the metal ion in heme systems in solution

Single-crystal magnetic study on ferromagnetic manganese(II) phthalocyaninate

A magnetic study has been carried out in the temperature range 1.2 - 25 K and magnetic field range 0 to 50 kOe on single crystals of mangangese(II) phthalocyanine. At higher temperatures the magnetic properties of manganese(II) phthalocyanine exhibit chain-like characteristics which may be understood in terms of ferromagnetic Heisenberg intrachain exchange of S = 3/2 ions with a weak antiferromagnetic interchain interaction. In the ordered state, Tc = 8.3 K, MnPc is a canted ferromagnet with easy axes of magnetization being along X, and X3 directions. A zero-field splitting of the single ion A2 state of the manganese(If) ion gives rise to canted ferromagnetism which does not show complete saturation at the high field range of these experiments (50 kOe). The spin structure of manganese(II) phthalocyanine at low temperature is discussed

Stereochemical structure and biochemical activity of heme proteins

A correlation between the stereochemical geometry around the metal ion in heme enzymes and their catalytic properties is attempted. It is shown that spin-lattice relaxation time measurement of bulk water in the enzyme solution gives accurate information as to the presence or absence of water molecule at the sixth coordination site of the heme iron. The rate of formation of compound I, an intermediate in the catalytic cycle of the enzymes, is shown to be directly related to the nature of the sixth coordination site of the heme

Substrate modulates compound I formation in peroxide shunt pathway of Pseudomonas putida cytochrome P450<SUB>cam</SUB>

The active oxygenating intermediate, a ferryl-oxo-(II) porphyrin cation radical (compound I), in substrate-bound cytochrome P450cam (P450cam) has eluded detection and kinetic analysis for several decades. Upon rapid mixing of peroxides-H2O2 and m-CPBA with substrate-bound forms of P450cam, we observed an intermediate with spectral features characteristic of compound I. Unlike in H2O2, kinetic investigation on the reaction of m-CPBA with various substrate (camphor, adamantone, and norcamphor)-bound P450cam and its Y96A mutant shows a preferential binding of the aromatic end group of m-CPBA to the active-site of the enzyme and modulation of compound I formation by the local environment of heme active-site. The results presented in this paper describe the importance of heme environment in modulating formation of compound I, and form the first kinetic analysis of this intermediate in the peroxide shunt pathway of substrate-bound P450cam

Aggregation in five-coordinate high-spin natural hemins: determination of solution structure by proton NMR

1H NMR measurements (at 500 MHz) of nuclear spin-spin relaxation time T2 (from NMR line width) at different temperatures are reported for aggregates of several five-coordinate high-spin iron(III) complexes of proto-, deutero-, and coproporphyrins in solution and are utilized to determine their solution structure. Extensive aggregation of these complexes in solution is observed, and the dominant form of the aggregates is shown to be dimers. The degree of aggregation for these iron(III) porphyrins follows the order proto- &#187; deutero- &gt; copro-. The line width of the heme methyl resonances was analyzed by using a nonlinear least-squares fit program working in finite difference algorithm. The values of T2 were used to determine the structural details of the dimer

Role of protein and substrate dynamics in catalysis by Pseudomonas putida cytochrome P450<SUB>cam</SUB>

The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450cam (P450cam) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450cam, and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (k2) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450cam suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450cam active site. We monitored substrate dynamics in the active site region of P450cam by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450cam indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450cam complexes. The present study shows that P450cam local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450cam heme pocket

Kinetics and thermodynamics of the molecular mechanism of the reductive half-reaction of xanthine oxidase

The kinetics and thermodynamics of the reductive half-reaction of xanthine oxidase with xanthine as substrate have been investigated by stopped-flow kinetic measurements. The temperature dependence of the steady-state and transient kinetics of the reductive half-reaction reveals the existence of at least three molecular intermediates during this half-reaction. All the microscopic rate constants and the thermodynamic activation parameters of the elementary steps of the reductive half-reaction have been determined for the first time. The microscopic rate constants and the thermodynamic activation parameters of the individual steps show wide variations in their magnitudes. The present work provides the most detailed and incisive description of the reaction of xanthine oxidase with its physiological substrate xanthine

Altered redox affinity of xanthine oxidase active sites by copper(II) ions

The interaction of Cu2+ ion with the redox centres of xanthine oxidase (XO) has been investigated using optical difference spectroscopic measurements. Anaerobic enzyme-reduction experiments using controlled and excess substrates (xanthine and NADH) have been performed to investigate the perturbation of XO active sites in the presence of Cu2+ ions. The results indicate an overall alteration in the redox affinities (i.e. affinity to accept electrons) of the active sites of XO by Cu2+ ion

Kinetic studies of the two-step reactions of H<SUB>2</SUB>O<SUB>2</SUB> with manganese-reconstituted myoglobin

Kinetics and thermodynamics of the reaction of manganese-reconstituted myoglobin (MnMb) with hydrogen peroxide have been investigated by the stopped-flow kinetic technique. The results show evidence of two-step formation of peroxide compound in MnMb. Detailed kinetic investigation provides the complete reaction mechanism of the formation of the peroxide compound. It is observed that the formation of the peroxide compound involves an equilibrium binding step with MnMb and H2O2. The H2O2 bound complex of MnMb undergoes irreversible transformation to the peroxide compound, MnMb-I. The microscopic rate constants, involved during these elementary transformation reactions, have been determined. The detailed thermodynamic investigation of the elementary transformation enables us to construct their energy diagram