Crystal structure of rat liver NADPH-cyctochrome P-450 reductase

Abstract

The t1avoprotein NADH peroxidase (NPX) from Streptococcus .fi1ecalis represents one of two known flavin-dependent hydroperoxidases. Its flavin-linked function is chemically similar to flavoprotein disulfide reductases such as glutathioi1e reductase. However. in NPX. an unusuaL stabilized cysteine-sulfenic acid (Cys-SOH) is the redox center. in contrast to the redox active disulfide group of glutathione reductase. Indirect evidence for the ~ existence of the Cys-SOH moeity resulted from metabolic labeling studies and FAB-mass spectrometric analysis of an active-site cysteinyl peptide (Poole and Claiborne, 1989). However. the initial x-ray crystal structure of the wild-type peroxidase refined at 2.16 A resolution revealed that the Cys42 had been oxidized to the inactive sulfonic-acid form (Cys-S03H) (Stehle et. al, 1991 ).In order establish the existence of the Cys-SOH form of the NPX, the x-ray crystal structure of the peroxidase with its native Cys42-sulfenic acid redox center was determined. To obtain the native, unoxidized enzyme, a strategy combining reduced exposure to ambient oxygen and data collection at -l60'C was used. The structure containing the native. redox active site conclusively establish the existence of the Cys-42 sulfenic acid moiety as the functional nonflavin redox center of the peroxidase and provide the first structure for any naturally-occurring protein-sulfenic acid. 78284.NADPH-cytochrome P-450 reductase (CPR) catalyzes the transfer of electrons from NADPH to all known microsomal cytochromes P450. CPR consists of a large 70 kDa cytoplasmic domain that contains both FMN and FAD as well as a NADPH binding domain, and a small, ~50 residue hydrophobic N-terminal domain responsible for anchming the protein to the endoplasmic reticulum or nuclear envelope of most eukaryotic cells. Electron transfer proceeds from NADPH to FAD to FMN and finally to the heme of cytochmme P-450.We have crystallized the cloned cytoplasmic domain of rat CPR and have collected a native data set to 2.6 on a Rigaku R-AXIS image plate detector with Rsvm = 7.1 %. The crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit cell parameters a= 103.28, b= 116.18 and c= 119.77 A. The V m is 2.6A3/dalton, with two molecules per asymmetric unit. The two molecules are related by a local two fold a;\is which is almost parallel to c.The structure has been solved to 3.0 by MIR, with four heavy atom derivatives and a figure of merit was 0.595. The phases were extended and improved by using solvent flattening and local-twofold averaging to 3 A. The model was built by using the TURBO package and refined by XPLOR. The current R factor is 27.8%. Further refinement is in progress.The copper-containing amine oxidase from the yeast Hansenula polvmOJ]Jlw has been solved by molecular replacement. It is a ubiquitous dimeric enzyme that catalyze the oxidative deamination of primmy amines by moleculm-oxygen to the cotTesponding aldehydes, ammonium and hydrogen peroxide. Copper m11ine oxidases are a novel gmup of quinoenzymes tha...