Cytochrome P450 purified from Fusarium oxysporum (P450nor) is a unique heme enzyme that catalyzes the reduction of nitric oxide to nitrous oxide with electrons directly transferred from NADH (2NO + NADH + H+--> N2O + H2O + NAD+). We studied the reaction of P450nor with NO and NADH using stopped-flow rapid scan and low temperature spectroscopic methods. The NO ligand can bind to the ferric enzyme to form the stable NO bound complex, P450nor(Fe3+NO). Reduction of P450nor(Fe3+NO) with NADH yielded an intermediate, which transiently formed (tau = approximately 100 ms) and spontaneously decomposed to the Fe3+ state. The optical absorption spectrum of the intermediate was different from that of P450nor(Fe2+NO), which was formed by either a one-electron reduction of P450nor(Fe3+NO) with Na2S2O4 or NO binding to P450nor(Fe2+). On the basis of these observations, we suggested that the intermediate is presumably a two-electron reduced product of P450nor(Fe3+NO) by NADH, formally the (Fe3+NO)2-complex. We determined the rate constants of these reactions at 10 degrees C for the NO binding to P450nor(Fe3+) (2.6 x 10(7) M-1 s-1), the NADH reduction of P450nor(Fe3+NO) (0.9 x 10(6) M-1 s-1), and the spontaneous decomposition of the intermediate (0.027 s-1). In these kinetic measurements, it was found that the former two processes are fast enough, while the latter is extremely slow, compared with the fast turnover of the catalytic reaction (1200 s-1 at 10 degrees C), which we measured by monitoring the NADH consumption. Therefore, we suggested that in the catalytic cycle, decomposition of the intermediate is fairly accelerated by free NO, resulting in such a fast turnover. On the basis of several lines of the spectroscopic and the kinetic evidence, we proposed a possible mechanism of the NO reduction by P450nor.
BackgroundMHC class I (MHCI) molecules are the key presenters of peptides generated through the intracellular pathway to CD8-positive T-cells. In fish, MHCI genes were first identified in the early 1990′s, but we still know little about their functional relevance. The expansion and presumed sub-functionalization of cod MHCI and access to many published fish genome sequences provide us with the incentive to undertake a comprehensive study of deduced teleost fish MHCI molecules.ResultsWe expand the known MHCI lineages in teleosts to five with identification of a new lineage defined as P. The two lineages U and Z, which both include presumed peptide binding classical/typical molecules besides more derived molecules, are present in all teleosts analyzed. The U lineage displays two modes of evolution, most pronouncedly observed in classical-type alpha 1 domains; cod and stickleback have expanded on one of at least eight ancient alpha 1 domain lineages as opposed to many other teleosts that preserved a number of these ancient lineages. The Z lineage comes in a typical format present in all analyzed ray-finned fish species as well as lungfish. The typical Z format displays an unprecedented conservation of almost all 37 residues predicted to make up the peptide binding groove. However, also co-existing atypical Z sub-lineage molecules, which lost the presumed peptide binding motif, are found in some fish like carps and cavefish. The remaining three lineages, L, S and P, are not predicted to bind peptides and are lost in some species.ConclusionsMuch like tetrapods, teleosts have polymorphic classical peptide binding MHCI molecules, a number of classical-similar non-classical MHCI molecules, and some members of more diverged MHCI lineages. Different from tetrapods, however, is that in some teleosts the classical MHCI polymorphism incorporates multiple ancient MHCI domain lineages. Also different from tetrapods is that teleosts have typical Z molecules, in which the residues that presumably form the peptide binding groove have been almost completely conserved for over 400 million years. The reasons for the uniquely teleost evolution modes of peptide binding MHCI molecules remain an enigma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0309-1) contains supplementary material, which is available to authorized users.
Nitric oxide reductase from the denitrifying fungus Fusarium oxysporum catalyzes the reduction of NO to N 2 O [Nakahara, K., et al. J. Biol. Chem. 1993, 268, 8350-8355]. Since this enzyme belongs to the cytochrome P450 superfamily [Kizawa, H., et al. J. Biol. Chem. 1991, 266, 10632-10637], it is called cytochrome P450nor (P450nor), but does not exhibit monooxygenation activity. In the present study, we examine the coordination structure of the heme iron in P450nor in the ferric-NO form by using infrared, resonance Raman, and X-ray absorption (EXAFS ) extended X-ray absorption fine structure) spectroscopies, since the ferric-NO complex is a first intermediate in the NO reduction cycle by P450nor [Shiro, Y, et al J. Biol. Chem. 1995, 270, 1617-1623. We compared the data obtained with those for the d-camphor-bound form of Pseudomonas putida camphor hydroxylase cytochrome P450cam (P450cam), a typical model of the monooxygenase. From the vibrational spectroscopic measurements, we found that the Fe-bound N-O stretching frequency (ν(N-O)) occurred at 1851 cm -1 and the Fe-NO stretching frequency (ν(Fe-NO)) at 530 cm -1 for P450nor, while those at 1806 and 522 cm -1 were observed for P450cam, respectively. The assignments were confirmed by the 15 NO substituting effect on these vibrational frequencies. These results indicated that NO binds to the ferric iron in P450nor stronger than in P450cam. Support for this was provided from the EXAFS study, which gave an Fe-N NO bond distance of 1.66 ( 0.02 Å for P450nor and 1.76 ( 0.02 Å for P450cam. These spectroscopic results suggest that, compared with P450cam, the lower steric hindrance and/or the difference in the electrostatic interactions of the ligand NO with its surroundings facilitates the donation of the NO 2pπ* electron to the iron 3dπ orbital, resulting in the strengthening of the Fe-NO and the N-O bonds of P450nor. The vibrational spectral observation of the ferrous-CO complex of P450nor supported this suggestion. This configuration can reduce the electron density on the NO ligand in P450nor, which is seemingly relevant to the NO reduction reactivity of P450nor.
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