Shinya Yoshikawa

Steady-state kinetics of oxidation of reduced ferredoxin with ferredoxin-nadp+ reductase

Abstract The electron-transfer reaction from dithionite-reduced ferredoxin to NADP+ catalyzed by Spirulina platensis ferredoxin NADP+ reductase (ferredoxin:NADP+ oxidoreductase, EC 1.18.1.2), was investigated at the initial steady state under anaerobic conditions. The initial velocity patterns and the inhibitory effect of oxidized ferredoxin suggested a reaction scheme in which reduced ferredoxin and NADP+ bound to the free enzyme sequentially to form a ternary complex. This mechanism is different from that (a ping-pong mechanism) proposed for the NADP+ reductase reaction based on the analysis of a widely used model reaction system using NADPH as an electron donor in NADP+ diaphorase assay system and in ferredoxindependent cytochrome c reducing assay system. The effects of pH and metallic cations in the physiological NADP+ reductase reaction designed in the present experiment also showed that the reaction was not simply the reverse of that observed in those model reactions.

Resonance raman spectra for catalytic intermediates of cytochrome c oxidase detected with a mixed flow transient apparatus

A novel technique was employed to collect resonance Raman spectra of an oxygenated intermediate of cytochrome c oxidase. Instead of laser pulses of high peak power, which may cause photodissociation, a continuous wave laser and a mixed flow apparatus were used. An intermediate formed within 450 microseconds after the reaction of cytochrome c oxidase with molecular oxygen could be detected. From the spectra it could be deduced that the most likely candidate for the intermediate would be a transient oxygenated species having the Fe2+ - O2 or Fe4+ = O heme in cytochrome a3 and the Fe2+ heme in cytochrome a.

Determination of the effects of Mg2+ on the O2 affinity of chlorocruorin by resonance Raman spectroscopy

Abstract Resonance Raman Spectra of deoxy- and oxychlorocruorin are reported for the first time. The oxy form was found to undergo extensive photodissociation upon illumination by laser light at 441/6 mm. Taking this advanatage, we determined the effect of Mg2+ on the O2 affinity of chlorocruorin. The oxygen affinity increased linearly with the Mg2+ concentration up to 5 mM, above which it was constant. The Fe(II)-histodine stretching Raman line of the deoxy form appeared at the frequency of the high-affinity form of the tetrameric hemoglobin and remained unlatered upon addition of Mg2+, despite a 3-fold increase in the O2 affinity at 5 mM Mg2+.

Mechanism of Dioxygen Reduction by Cytochrome c Oxidase as Studied by Time-Resolved Resonance Raman Spectroscopy

Time-resolved resonance Raman (TR3) spectroscopy has been applied to cytochrome c oxidase (CcO) to elucidate the mechanism of dioxygen reduction. Six oxygen isotope-sensitive Raman bands have been identified in the TR3 spectra. The “607-nm species” defined by difference absorption spectrum, which is referenced against the oxidized enzyme, is demonstrated to have an Fe=O heme, although it has long been believed to have an Fe-O-O-X (X=H or CuB) heme. The one-electron reduction of this Fe=O intermediate, which yields the oxoferryl intermediate, is demonstrated to be coupled with proton transfer in the protein. The mechanism of dioxygen reduction by CcO is discussed on the basis of the structures of the reaction intermediates.