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Escherichia coliflavorubredoxin (FlRd) belongs to the family of flavodiiron proteins (FDPs), microbial enzymes that are expressed to scavenge nitric oxide (NO) under anaerobic conditions. To degrade NO, FlRd has to be reduced by NADH via the FAD-binding protein flavorubredoxin reduc-tase, thus the kinetics of electron transfer along this pathway was investi-gated by stopped-flow absorption spectroscopy. | ỊFEBS Journal Kinetics of electron transfer from NADH to the Escherichia coli nitric oxide reductase flavorubredoxin Joao B. Vicente1 Francesca M. Scandurra2 Joao V. Rodrigues1 Maurizio Brunori2 Paolo Sarti2 Miguel Teixeira1 and Alessandro Giuffre2 1 Institute de Tecnologia Quimica e Biologica Universidade Nova de Lisboa Oeiras Portugal 2 Department of BiochemicalSciences CNR Institute of Molecular Biology and Pathology and Istituto Pasteur - Fondazione Cenci Bolognetti University of Rome La Sapienza Italy Keywords flavodiiron proteins microbialNO detoxification NADH rubredoxin oxidoreductase nitrosative stress time-resolved spectroscopy Correspondence A. Giuffre Istituto di Biologia e Patologia Molecolari delConsiglio Nazionale delle Ricerche c o Dipartimento di Scienze Biochimiche A. Rossi Fanelli University di Roma La Sapienza Piazzale Aldo Moro 5 I-00185 Roma Italia Fax 39 06 4440062 Tel 39 06 49910944 E-mail alessandro.giuffre@uniroma1.it Received 22 September 2006 revised 20 November 2006 accepted 21 November 2006 Escherichia coli flavorubredoxin FlRd belongs to the family of flavodiiron proteins FDPs microbial enzymes that are expressed to scavenge nitric oxide NO under anaerobic conditions. To degrade NO FlRd has to be reduced by NADH via the FAD-binding protein flavorubredoxin reductase thus the kinetics of electron transfer along this pathway was investigated by stopped-flow absorption spectroscopy. We found that NADH but not NADPH quickly reduces the FlRd-reductase k 5.5 2.2 X 106 M-1-s-1 at 5 C with a limiting rate of 255 17 s-1. The reductase in turn quickly reduces the rubredoxin Rd center of FlRd as assessed at 5 C working with the native FlRd enzyme k 2.4 0.1 X 106 M-1-s-1 and with its isolated Rd-domain k w 1 X 107 M-1-s-1 in both cases the reaction was found to be dependent on pH and ionic strength. In FlRd the fast reduction of the Rd center occurs synchronously with the formation of flavin mononucleotide semiquinone. Our data provide evidence .
